The movie begins where RE4 left off, with Alice (Milla Jovovich) on a boat full of zombie survivors being attacked by Umbrella corporation troops led by a brainwashed Jill Valentine (Sienna Guillory). Alice is knocked off the boat by an explosion and wakes up in an Umbrella cell in a secret location. Scenes are also shown of Milla Jovovich as a house wife, married to Oded Fehr (actor who played Carlos in 2 and 3) and with a hearing impaired daughter, Becky, when a zombie outbreak hits their suburban home. Milla and the daughter try to escape with the help of a local woman played by Michelle Rodriguez.
Back in the prison Alice is broken out by Ada Wong (Bingbing Li) working for Wesker (Shawn Roberts). Their goal is to escape the underwater prison run by the Red Queen with the help of a team coming down from the surface including Luther West from RE4. Ada and Alice fight through viral testing centers where the Red Queen releases biohazards in the forms of monsters from previous movies. Upon reaching suburbia, it is revealed in order to properly test viral and human reactions the Red Queen has been filling the testing centers with clones of various characters imprinted with false memories and then turned loose. The previous Milla housewife clone is dead, but Alice rescues the daughter before meeting up with Jill Valentine and the security team formed of previous people from earlier RE movies. Ada sends Alice and Becky off while she fights the security team. Alice and Becky are reunited with a terrified Michelle Rodriguez clone who watches Becky while Alice looks for help.
In the meantime, the outside team is trapped in a Moscow simulation until Alice shows up and rescues the surviving members. They reclaim Becky and Michelle and resume fighting their way to an escape elevator trying to beat a bomb set to destroy the facilities. The security team shows up with Ada as a hostage. Also, a previous super monster shows up. In the ensuing fire fight, most of the characters are killed with Alice, the daughter, and 2 of the rescue team escaping in the elevator.
Once at the surface, Jill, the Rain clone (Michelle Rodriguez) and Ada appear, having escaped in a sub. Milla fights Jill and Rain who has injected herself with a super virus. Milla manages to remove the mind control device from Jill who then helps her defeat Rain (who has killed Luther during the fight) by shooting the ice out from under her causing her to fall into the zombie filled water. Helicopters rescue the survivors who are flown to meet Wesker in an apocalyptic DC and are told that the Red Queen is trying to wipe out humanity. Wesker declares Alice to be humanity's last hope and injects her with t-virus before the camera pans out to show the entire compound surrounded by an army of zombies including what appears to be zombie dragons.