Alice (Milla Jovovich) wakes up disoriented in a mansion. Wandering through its halls she overcomes several obstacles, including a deadly laser beam and a giant blade that falls from the ceiling. However, she is eventually killed by a bounding mine. Her body is dumped into a pit filled with dozens of Alice copies and is revealed to be a clone. The camera zooms out to show a shack camouflaging the facility's above-ground entrance - with the rest of the facility located underground, surrounded by a high fence and thousands of zombies. Despite the best efforts of the bio-tech company Umbrella Corporation to cover up the contamination of The Hive and the release of the T-virus to the surface, going so far as to authorize the bombing of Raccoon City, the T-virus has spread around the world. As the virus spread, it affected not only humans, but other animals as well, and the environment deteriorated, turning cities into desolate landscapes and the wilderness into wastelands. The real Alice (from whom the clones are made) wanders the wastelands of the Southwestern U.S. She responds to a group asking for help on the radio. It turns out that they are bandits who first attempt to rape her and then have her killed by infected dogs, only for her to unleash them unto her captors. On her travels, she finds a dead man's diary which states that there is an uninfected area in Alaska.
Meanwhile, Dr. Alexander Isaacs (Iain Glen), former head of Project Alice and the head of the North American Umbrella facility, operated in Nevada, considers Alice's recapture to be a top priority, since she has the ability to bond with the T-virus and not suffer mutation. He tries to persuade the Umbrella board, operating via holograms from the worldwide Umbrella facility, that he can use her blood to develop a permanent cure as well as tame the infected zombies. The new Umbrella chairman Albert Wesker (Jason O'Mara) orders Isaacs to use the clones to create a cure, and he refuses to authorize Alice's recapture until they are informed of her location.
Elsewhere, a convoy of survivors led by Claire Redfield (Ali Larter) travels across the country in search of supplies. The convoy includes Raccoon City survivors Carlos Oliveira (Oded Fehr) and L.J. (Mike Epps), along with new survivors K-Mart (Spencer Locke), Mikey (Christopher Egan), Chase (Linden Ashby), Otto (Joe Hursley) and Nurse Betty (Ashanti Douglas), among others.
While searching a motel for supplies, L.J. is bitten by a zombie, which he keeps secret. The next morning the convoy is attacked by crows that had been feeding on the infected. The crows almost overwhelm the convoy, killing Nurse Betty and Otto and taking out the flamethrower operator, causing the weapon to careen out of control. Alice finally appears and saves Carlos and the others from a fiery death, using her telekinesis to fill the sky with fire, killing the crows and leaving her unconscious. After recovering, Alice is introduced to Claire and gives her the diary she found, and she and Carlos then convince Claire to take her convoy to Alaska.
Dr. Isaacs' attempts to domesticate the infected have led to a new zombie breed that remembers basic things, such as how to use phones and cameras. Wesker's security officer, Captain Alexander Slater (Matthew Marsden), is adamant about Isaacs' disregard for Umbrella's regulations. Wesker, in private, informs Slater to keep watch on Isaacs' actions, and tells Slater that if Issacs disobeys orders, Slater should kill Isaacs.
Umbrella triangulates Alice's location based on the signal sent out by her telekinetic abilities. Dr. Isaacs has a crate of his new zombies sent to ambush the convoy, against Wesker's specific orders. L.J. succumbs to his infection and bites Carlos. Most of the convoy, including Mikey and Chase, are killed in the ambush. Umbrella tries to shut Alice down remotely, but she breaks free from their programming and continues to fight. She finds Isaacs at the scene, and he becomes infected as he flees via helicopter. Alice and K-Mart use Isaacs' computer to track the helicopter's flight path, leading them to Umbrella's underground location.
The convoy heads over to the site, which is surrounded by thousands of zombies around the area. Carlos sacrifices himself by plowing his truck into the zombie mob and blowing them all up, giving Alice and Claire time to load survivors into the helicopter. Alice decides to stay behind. Ordered by Wesker to terminate Isaacs, Slater arrives with a group of commandos and finds the scientist locked in his lab, injecting himself with massive doses of anti-virus in an attempt to counteract the infection. Slater reveals that his employment is terminated and shoots him, triggering his mutation into a monstrous Tyrant. Despite retaining his intelligence and thought, Isaacs kills Slater and all the facility employees, but is confined to the lab's lower levels.
Entering the facility, Alice meets a holograph 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, defending the Red Queen's actions as well. She then tells Alice about Dr. Isaacs. Alice agrees to deal with him. On her way to the lab's lower levels, Alice discovers one of her clones, still in development. The clone awakens, but seems to die from shock upon seeing Alice. Alice finds a replica of the mansion she lived in and finds Isaacs, who fights her with equal strength and telekinesis. Finding themselves in a replica of The Hive's laser corridor, Alice awaits the grid's arrival, watching Isaacs getting sliced into cubes. Just as Alice is about to meet the same fate, the system is deactivated by her clone, which has inexplicably revived.
Later, in Tokyo, Japan, Wesker, who is the head of the Tokyo Umbrella facility, informs the rest of the Umbrella chairmen (all holograms) that the North American facility has been lost. Alice's hologram makes an appearance, declaring that she is coming for him, and she's bringing some of her "friends." Standing beside her clone, Alice looks out onto a huge number of pods containing her developing clones.