The film begins showing a clone of Alice (Milla Jovovich) waking at the mansion, in a scene that is almost identical to the one in Resident Evil. Alice moves through obstacles including the laser room seen in the first film, though she is eventually killed by an Umbrella weapons system. Her dead body is taken by three scientists, including Dr. Isaacs (Iain Glen), and dumped into a pit outside the Umbrella facility. The pit is shown to be filled with numerous Alice Clones. The camera zooms out from the facilities main entrance (the remaining facility being underground) and shows hundreds of zombies trying to enter the facility, which is surrounded by barbed wire fence.
Five years after Resident Evil: Apocalypse, Alice is now alone in the desert wastelands of the remains of the United States. She has superhuman strength and psionic abilities gained from Umbrella. Dr. Sam Isaacs considers re-capturing Alice a top priority because of her ability to bond with the T-Virus, and attempts to persuade the rest of the Umbrella board that he can use her blood to develop a permanent cure, as well as tame the infected. Umbrella chairman Albert Wesker (Jason O'Mara) orders Isaacs to create a cure using clones of Alice created from the blood of the original and refuses to authorize manpower to recover Alice until they are 100% sure of her location.
Meanwhile, Claire Redfield's (Ali Larter) convoy, which includes Alice's fellow Raccoon City survivors Carlos Olivera (Oded Fehr) and L.J. (Mike Epps), along with new survivors K-Mart (Spencer Locke), Mikey (Christopher Egan), Chase (Linden Ashby) and Nurse Betty (Ashanti Douglas), are attempting to survive. While searching a small motel for supplies, L.J. is bitten. The next morning the convoy is attacked by infected crows. The crows very nearly overpower the convoy, killing Nurse Betty and taking out the operator of the pintle-mounted flamethrower causing the weapon to careen out of control. Alice appears to save Carlos and a fellow survivor from a fiery death, using her mind powers on the flamethrower she covers the sky with a blanket of fire killing the plague of infected crows. Alice is introduced to Claire, and gives Claire a diary she found in a deserted gas station. The diary claims of an infection-free population living in Alaska. Alice and Carlos convince Claire and the convoy to head to Alaska. The other survivors agree to head for Las Vegas to obtain supplies before the trek to Alaska.
Dr. Isaacs' attempts to domesticate the infected have led to a new breed of zombie but Wesker's subordinate, Captain Slater (Matthew Marsden), is adamant about keeping Isaacs from disregarding Umbrella corporate regulations. After Isaacs explains himself, Slater admits that these Super Zombies (the Crimson Heads) could be useful in re-capturing "Project Alice", but that Isaacs is too out of control.
The Umbrella corporation is able to triangulate Alice's location based on the use of her psionic powers, and Dr. Isaacs against Chairman Wesker's orders has a crate full of his newly created zombies transported to ambush the convoy. Carlos becomes infected by L.J., and most of those in the convoy, including Chase and Mikey, are killed in the ensuing battle. Umbrella attempts to shut Alice down; however, she breaks free of their programming and fights on. She locates Isaacs at the scene, and he is infected by a Crimson Head as he attempts to flee. Alice and K-Mart enter the Umbrella tent and use Isaacs' computer to track the helicopter's flight path, thus learning Umbrella's underground location.
The convoy of survivors find the location of Umbrella's Las Vegas facility surrounded by hundreds of zombies. Carlos' infection has gone beyond a cure, and he sacrifices himself for the convoy by plowing through the facility before setting off a dynamite charge, giving Alice and Claire time to load the survivors onto the helicopter. Alice decides to stay behind and finish the mission. Dr. Isaacs has been locked in his lab. Ordered by Wesker to "liquidate" Isaacs, Slater finds him at his lab desk, injecting himself with massive doses of Anti-Virus in an attempt to counter-act the mutation and the T-virus. Slater shoots Dr. Isaacs, triggering mutation into a Tyrant. Isaacs goes on a rampage, killing all the employees in the facility, but the White Queen manages to confine Isaacs in the lower levels of the lab.
Entering the facility, Alice meets a holographic representation of the Red Queen's sister AI, the White Queen (Madeline Carroll). The White Queen informs Alice that her blood is the cure to the T-Virus. Then she informs Alice of her current problem with Dr. Isaacs, and Alice agrees to deal with him. On her way to the lab's lower levels, Alice comes across a clone of herself still in a vegetative state. The clone awakens, but seemingly dies from shock. A battle between Isaacs and Alice spills into a replica of the laser corridor from The Hive, the laser grid activates and slices Isaacs into pieces, but just as Alice is about to meet the same fate, the lasers are deactivated by the clone of Alice who woke up in the nick of time.
Later, in Tokyo, Japan, Wesker informs the rest of the Umbrella board that the North American branch has been lost, and he will now control further scientific experimentation from Japan. Suddenly, Alice makes an appearance at the board meeting from the North American facility, declaring that she and her friends are coming for them now. Standing beside the clone of herself, Alice looks onto the rows of pods containing other clones who are awakening.