A group of people are taking a tour of the Aldridge mansion in New York City. The tour guide Garrett (Zach Woods) tells the group of Gertrude Aldridge, a disturbed woman that was kept locked in the basement by her father. A candlestick falls over on the table near the basement to creep the group out, but it's just part of a trick Garrett played. What he doesn't see is a device beneath the nightstand that is flickering and vibrating.
As he is closing up shop, Garrett hears a noise near the basement. The door begins shaking, and he runs for it. Garrett tries to run out of the mansion, but the doorknob has been heated. He tries to break out a window with a stool, only for the stool to be thrown back. Garrett runs back toward the basement. The floor is filling up with green slime, and the stairs break apart. Garrett holds on as he sees a ghost rise up from the floor.
At Columbia University, Dr. Erin Gilbert (Kristen Wiig) is approached by Ed Mulgrave (Ed Begley, Jr.), a publisher who has a book that Erin wrote some years ago on the existence of ghosts. She's not proud of having written it, and she's even less than pleased to learn it has resurfaced. Erin knows it was done by her former friend and colleague Abby Yates (Melissa McCarthy). Ed approaches Erin because of the haunting at the Aldridge mansion and asks her to look in on it. As Erin looks up Abby's current workplace, she learns from the university's dean Harold Filmore (Charles Dance) that she is up for tenure, so she does what she can to hide knowledge of the book.
Erin finds Abby in her lab with her new work partner Jillian Holtzmann (Kate McKinnon), a brilliant engineer who is also a bit nutty. Erin and Abby are still upset with each other over the book, and Erin demands Abby take the book off Amazon. Erin then brings up the Aldridge mansion haunting, inspiring Abby and Holtzmann to spring into action, with Erin reluctantly joining them.
The three ladies arrive at the mansion with some equipment. The basement has been locked tight. Erin steps on some slime. Suddenly, the three notice that the basement door is now open and emitting an eerie blue glow. They are then face-to-face with the ghost of Gertrude Aldridge. Erin approaches her calmly, but Gertrude's face turns demonic and she spews ectoplasmic bile all over Erin. The ghost flies out of the building. The ladies record a video with Erin proclaiming that ghosts are real. Unfortunately, this falls into Filmore's hands, and he fires Erin for being a bad representation of the school.
Now that Erin is jobless, she decides to join Abby and Holtzmann in their research. Abby and Holtzmann also lose their job after talking to the dean (who happens to be a dick), so they steal equipment from the school's lab and decide to open up their own facility.
We meet MTA worker Patty Tolan (Leslie Jones). She is approached by a weird guy named Rowan North (Neil Casey). He rambles on about a "fourth cataclysm", which Patty pays no mind to. She then has to go into the tunnel to check something out. Behind her, Rowan plants another device similar to the one at the mansion. Patty then sees a ghost in a prison suit. She runs like hell away from it.
Erin, Abby, and Holtzmann set up shop in a new location above a Chinese food restaurant as the "Department of Metaphysical Examination". A young man named Kevin Beckman (Chris Hemsworth) shows up for a receptionist job. Erin thinks he's hot, but during the interview, Kevin proves to be really dumb. Still, they hire him on the spot. The ladies are then visited by Patty, who mentions the ghost she spotted in the subway.
Patty leads the ladies to the tunnel where she spotted the ghost. Holtzmann brings along some equipment she built to catch the ghost. They see the same ghost that Patty saw hovering above them. They record the ghost with their HD cams while Erin is given a proton laser to capture it. The laser isn't strong enough to hold the ghost down, and the train is coming in fast. The ladies run to safety as the train hits the ghost, causing them to get splattered with ectoplasm slime while the ghost is stuck on the train.
The video of the ghost goes viral, and the media dubs the team "Ghostbusters", to their chagrin. Famed de-bunker Martin Heiss (Bill Murray) goes on record to dismiss the subway ghost as fake. Patty decides to join the team since she knows how to get around the city, and she provides the team with a vehicle for carrying equipment. It's a hearse that she borrowed from her uncle, which only Holtzmann seems to enjoy, and it becomes the new Ecto-1.
Hotlzmann shows off some new equipment she made to catch ghosts, including a new laser gun, a grenade, and a proton glove. It's all untested, so most of it turns out to be dangerous, but the team thinks it's all cool.
The team is called to investigate a ghostly sighting at a musical theater. Rowan is seen planting another device in the theater. Abby's specter detector (not the actual name of the device) goes off near the dressing rooms. Patty is chased by a mannequin possessed by a ghost. The Ghostbusters hit the mannequin with their proton lasers, releasing a huge flying ghost monster into the theater. They follow it up to the stage where a band called Beasts of Mayhem are performing. The ghost flies out, making everyone think it's part of the show until the Ghostbusters rush the stage and try to hit it with their lasers. Abby stage-dives and goes for it, and Patty follows, but nobody catches her. She gets up and has the ghost perched on her shoulders. The other three wait for Patty to be still as they hit it with their lasers. Holtzmann pulls out a trap and has the ghost sucked into it. Everyone cheers the team on, while Ozzy Osbourne is backstage and thinks he's having a "flashback".
The team celebrates their first capture with some pizza. Abby and Erin discuss how they were friends since high school. Erin also mentions how when she was a child, the ghost of her mean neighbor would stand at the foot of her bed every day for a year, and nobody believed her when she said it. They are then visited by Martin Heiss so he can prove that the team is faking their capture. Erin gets annoyed and decides to prove Heiss right by bringing out the trap and releasing the ghost. The ghost flies out and pushes Heiss out a window as it escapes into the city.
After the police arrive, the team is escorted by Agents Hawkins (Michael Kenneth Williams) and Rourke (Matt Walsh) to meet with Mayor Bradley (Andy Garcia) and his assistant Jennifer Lynch (Cecily Strong). While they are thankful for the team's capture of the ghost, they want them to say they are frauds so as to not cause a stir in the city.
When the team is alerted to a new ghost sighting, Erin realizes there is a pattern to all the appearances in the city. It is discovered that all the devices found are along leylines that intersect at the Mercado Hotel. They find a picture of the staff, and Patty recognizes Rowan among them, knowing he is their guy. The team heads to the hotel and finds Rowan in the basement with a device he built in which he plans to release a horde of ghosts upon the city as payback for being constantly mistreated. Abby tries to convince Rowan to stop his plans until she mentions the police are on their way. Rowan then grabs two pillars and electrocutes himself before the police arrive. The basement is then closed off, but the specter detector is left behind and goes haywire.
At home, Erin reads through a copy of her book that Rowan was keeping to orchestrate his plan. She looks through the drawings and sees that he had always planned to electrocute himself so that he can become a ghost and eventually bring about the Fourth Cataclysm to lead the ghosts and terrorize the world.
At headquarters, Abby hears a knocking at the door, but no one is there. There is a rattling on the sink's pipe, and she sees a green glow in the drain. Rowan's ghost then possesses Abby. She starts breaking Holtzmann's equipment and then grabs Holtzmann by the throat to dangle her out a window. Patty intervenes and rescues Holtzmann before smacking Rowan out of Abby. Moments later, Kevin pulls up on his motorcycle, and Rowan possesses him. He rides off back to the hotel.
Erin runs to a restaurant where she finds Mayor Bradley and Lynch eating. She frantically tries to warn them to evacuate the city, but she comes off as crazy, so she is taken away.
Rowan in Kevin's body makes it to the hotel basement and attacks the two guards standing outside. He manages to break the barriers keeping the ghosts trapped to allow them to fly free, beginning the ghost invasion upon the city. A portal opens in the sky, releasing more and more ghosts upon the city. Erin tries to get a cab, but the cabbie (Dan Aykroyd) dismisses her and drives off.
Abby, Holtzmann, and Patty gear up and head off the fight ghosts. They run into none other than Slimer (Adam Ray) the green ghost eating from a hot dog cart. He steals the Ecto-1 and drives off. Abby and Patty get ready to shoot at the car, but Holtzmann says the equipment on top of the car is basically a nuke. They then spot a bunch of ghosts that look like giant parade floats, which they start popping with their proton packs. A float in the shape of the Stay-Puft Marshmallow Man shows up and presses down on the three, rendering them immobile. Erin saves them by popping Stay-Puft with a Swiss army knife that Holtzmann gave her. The team then faces off against dozens of ghosts, including Gertrude, the subway ghost, and the theater ghost.
The Ghostbusters head to the hotel to find a still-possessed Kevin having frozen the police and military. Rowan leaves Kevin's body and decides to take on a familiar, more "friendly" form - the Ghostbusters logo. However, he grows into a humongous beast that bursts out of the hotel and begins wreaking havoc on the city. As they see Slimer and other ghosts riding down the street, the team gets the idea to hit the Ecto-1's nuclear reactor into the portal to reverse it and eventually close it. They fire their lasers at the ghosts to have them steer into the portal before hitting the reactor. The plan works, and the ghosts start getting pulled back into the portal. Rowan holds on to two buildings, so the team hits him in the groin with their lasers to loosen his grip. He starts to get sucked in, but he grabs Abby and pulls her into the portal. Erin hooks herself up to a wire and jumps into the portal. She fires her laser at Rowan's hand to release Abby while he is pulled into the abyss. Holtzmann and Patty pull the two out, and they return with totally beached white hair.
Afterward, Mayor Bradley continues to deny the ghost problem, but he secretly gives the team funding to continue their work. They upgrade their headquarters to a firehouse (same as the original movie). Patty's uncle Bill (Ernie Hudson) then pulls in with a new hearse. He's not happy about what happened to the other one.
During the credits, we see the team being visited by Holtzmann's mentor Rebecca Gorin (Sigourney Weaver), who inspired her to be crazy. Patty then calls the team to the roof. The city's buildings are lit up to say "I Love GB". There is also a dedication to Harold Ramis.
After the credits, the team is doing some work while Patty is listening to a recording. Erin walks over to see what's up. Patty simply asks, "What's Zuul?"