Dr. James Marcus, the founder of the Umbrella
Corporation, had a daughter, Alicia, who was dying of progeria. Desperate
to save her, Marcus uses the T-virus on her and others with the
disease. After another child treated with the virus died and then reanimated as
a rabid zombie, Marcus tried to have the program shut down. His partner, Dr.
Alexander Isaacs, had Marcus killed by Albert Wesker, then adopted Alicia and
took over the Corporation.
After the events in Washington D.C., Alice awakens after being betrayed by Wesker. The Red Queen appears and tells Alice she has 48 hours to infiltrate the Hive, over 400 miles west on I-64 in Raccoon City. The Umbrella Corporation has developed an airborne anti-virus, which can kill every organism infected by the T-virus, but is waiting for the last few remaining humans to be wiped out.
Enroute to Raccoon City, Alice is captured by Isaacs, learning the "Isaacs" she previously killed was a clone. She escapes his convoy and reaches Raccoon City, where she is ambushed by a group of survivors: Doc, Abigail, Christian, Cobalt, Razor, and Claire Redfield, who survived the attack on Arcadia. Isaacs' convoy approaches, trailed by a horde of zombies. Alice and the group defeat the convoy, retrieve Isaacs' few human captives and burn the horde, though Cobalt is killed. Alice and crew enter the Hive entrance, at the bottom of the crater from the nuclear bomb previously dropped on the city. Wesker, in control of the Hive, releases mutated guard dogs, killing Christian and a freed captive.
The Red Queen appears to Alice and explains that her program is in conflict, as she can never hurt an Umbrella employee but also must value human life. She plays a video of Isaacs explaining to Umbrella's executives a plan to release the T-virus, cleansing the world of humanity; many of the rich and powerful, including the company executives, are stored in cryogenic capsules in the Hive, with the intention of rebuilding the world following the resulting apocalypse. The Red Queen warns Alice that someone in her group is helping Umbrella.
The group encounters several traps, killing Abigail and Razor. Alice and Doc plant bombs throughout the Hive, from the leftover equipment of the original team to investigate the Hive. Alice confronts the real, tech-upgraded, Isaacs. Doc is revealed to be Umbrella's spy, and Claire is captured by Wesker. A cryogenic capsule opens, releasing Alicia Marcus, Umbrella's co-owner, and Marcus' daughter. Isaacs reveals to Alice that she is actually a clone of Alicia, before going on to explain his plan to eliminate the pair and assume control of Umbrella. Alicia fires Wesker, allowing the Red Queen to crush his legs with a security door. Doc tries to shoot Alice, but his gun is empty — as Alice had earlier deduced his treachery — and Claire executes him. After giving Wesker a deadman's switch to the primed bombs, Alice and Claire pursue Isaacs while Alicia uploads a copy of her childhood memories.
Isaacs, Alice and Claire fight; Isaacs overpowers Claire and attacks Alice in the Laser Corridor, eventually defeating her, but she uses that moment to activate a grenade in his pocket. She escapes to the surface with the anti-virus, but Isaacs reboots and catches her before she can release it. Before he can kill her, the Isaacs clone (from the convoy) arrives and kills him, believing itself to be the original Isaacs, but the clone is then devoured by the undead. Alice releases the anti-virus, killing all of the undead around her before she passes out. Wesker simultaneously drops the deadman's switch, destroying himself, Alicia, the Hive, and the hibernating Umbrella elite.
Claire wakes Alice, who survived because the anti-virus killed only the T-virus within her body, not the healthy cells. The Red Queen uploads Alicia's childhood memories into Alice, granting her a childhood. Alice travels into Manhattan, saying that the anti-virus, carried only by the winds, will take years to reach all corners of the globe, and until it does, her mission is not finished.