The game is set in Raccoon City during a zombie apocalypse caused by an outbreak of the T-Virus that was created by pharmaceutical company Umbrella Corporation. On September 28, 1998, former Special Tactics And Rescue Service (S.T.A.R.S.) member Jill Valentine is attacked in her apartment by an Umbrella-created intelligent bioweapon known as Nemesis, who attempts to kill her and all remaining members of S.T.A.R.S.
Upon escaping her building, she meets up with fellow S.T.A.R.S. officer Brad Vickers, but Brad is bitten by a zombie and tells Jill to save herself. After another encounter with Nemesis, she is saved by Umbrella Biohazard Countermeasure Service (U.B.C.S.) mercenary Carlos Oliveira.
Carlos and his group of surviving U.B.C.S. mercenaries - Mikhail Victor, Tyrell Patrick, and Nicholai Ginovaef - have set up subway trains which they plan to use to evacuate civilians from the city.
After surviving several encounters with Nemesis, Jill manages to reactivate power to the subway, while Carlos and Tyrell remain in the city to search for Dr. Nathaniel Bard, an Umbrella scientist who might know how to make a vaccine for the T-virus and save the city.
As Jill, Nicholai, and Mikhail depart in the train, Mikhail expresses his suspicions towards Nicholai on how their platoon was ambushed by zombies. Nemesis suddenly attacks the train and kills the civilians; Nicholai locks the other two out, forcing them to defend themselves.
Nemesis grabs Mikhail, who sacrifices himself by detonating an explosive, causing the train to derail. Jill escapes as seemingly the only survivor of the crash.
Carlos and Tyrell travel to the city's police department, where they plan to find Bard in the S.T.A.R.S. office. The two witness R.P.D. Lieutenant Marvin Branagh being bitten by a zombified Brad, who is killed by Carlos shortly afterwards.
The two enter the S.T.A.R.S. office and communicate via video with Bard, who informs them that he is at a hospital. Meanwhile, as Tyrell traces Bard’s location, Jill escapes the wreckage of the derailed train only to be again pursued by Nemesis who is now heavily mutated.
Jill radios Carlos and engages the monster, managing to escape it, but only after it infects her with the T-virus. Jill falls unconscious and Carlos finds her roughly half a day later, taking her to Spencer Memorial Hospital — the location of Bard.
Carlos fights his way through the infested hospital only to find that Bard has been murdered. He views a video from Bard confessing that the T-virus was engineered by Umbrella, and despite Umbrella hiring him to develop the vaccine, Umbrella's board now wants to destroy it and eliminate all traces of the virus' existence.
Carlos retrieves the vaccine and administers it to Jill. Tyrell arrives at the hospital and they discover that the U.S. government plans to destroy Raccoon City in a missile strike to eradicate the T-Virus infestation. Carlos travels to the NEST 2 lab underneath the hospital to find more vaccines, while Tyrell tries to contact whoever he can to try and stop the missile strike.
Jill awakens on the day of the missile strike, October 1, and pursues Carlos to NEST 2. She encounters Nicholai, who is revealed to be a supervisor hired by an unknown contractor to sabotage Umbrella's efforts to hide their involvement and to observe and collect data from attacks of several bioweapons, including Nemesis.
Nemesis kills Tyrell and continues to pursue Jill throughout the lab. Jill manages to synthesize a vaccine, but an encounter with Nemesis prompts Nicholai to retrieve it for himself as he leaves her to fight the monster. Jill attempts to destroy Nemesis in a vat of solution designed to dispose of any biological waste, only for the NE-Alpha parasite and T-virus strain in Nemesis to prompt further mutation, resulting in a giant mass of flesh with tentacles.
Jill then uses a prototype railgun to finally eliminate Nemesis for good. At the hospital's helipad, Nicholai disarms Jill and destroys the vaccine, acknowledging that he doesn't care for the city's fate as long as he gets paid for sabotaging Umbrella. Carlos intervenes and restrains Nicholai for Jill to shoot.c When interrogated on who he works for, he offers to reveal the information and pay any price in exchange for his life.
Disgusted by his greed, Jill retrieves the destroyed vaccine case and escapes the city with Carlos via helicopter, leaving Nicholai behind. The city is destroyed by the missile strike, and Jill vows to take down Umbrella at all cost.
In a post-credit scene, Jill grabs the broken vaccine container from the table.