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It has been five years since the T-virus outbreak in Raccoon City. Despite Umbrella's best efforts to contain it, the lethal T-virus has spread across the globe, not only turning humans into zombies, but also causing rivers and lakes to dry up and reducing entire continents to deserts. Alice is on the run across the deserts that now cover the Western part of the United States. After facing a group of criminals that had managed to trap her using a fake radio emergency call, she finds a journal which details a route to Alaska; the state, being "isolated" as the journal says, might be virus-free.

Dr. Isaacs has been trying to reproduce/clone Alice's powers by creating clones of her, but none of them survives the tasks they are faced with (a mock-up of the Hive, the mansion and Raccoon General Hospital). He concludes to Wesker and the other Umbrella board members that he needs the original Project Alice in order to obtain her blood and hopefully find a mass cure for the virus. Wesker and the Umbrella board members thus made this together with the capture of Alice among their highest priorities. Using her clones' blood as a basis for the cure, he plans to domesticate the zombies with his newly developed serum harvested from Alice's blood. Initially, the enhanced zombie displayed signs of heightened intelligence, increased memory power and gentleness, but soon grew to be stronger, faster, and more vicious. Isaacs uses this to his advantage by secretly creating a bigger group of these 'super zombies', defying Wesker's orders.

At the same time, Carlos Olivera and L.J. have joined a convoy of survivors, consisting of K-Mart, Otto, Mikey, Chase Liam and Betty led by Claire Redfield, moving in search of supplies. While searching a motel, L.J. is bitten but doesn't tell anybody. At morning, the convoy is waken up by thousands of infected, hungry crows. When everything seems lost, Alice arrives and using her telekinesis manages to save the convoy. Despite her efforts, Betty, Otto and five others died in the attack.

She tells the group about her plan to Alaska, and they decide to follow it by popular vote. However, before trying the long journey they need supplies, and they decide to search the nearby Las Vegas, now in ruins. Meanwhile, Dr. Isaacs has been looking for Alice, hoping her DNA could be the source for a cure; he finally manages to track her by satellite, but only by a 62% confirmation. He later reports this to Wesker, who instead refuses to have Isaacs recapture Alice until the satellite confirmation hits 100%, but Isaacs is later revealed to have recorded Wesker's speech and edited it to make it appear as if Wesker ordered his second-in-command, Captain Alexander Slater, to send a force to capture Alice. Isaacs then sends a group of his newly created super-zombies to Las Vegas, both to test their ability and capture Alice.

The convoy is attacked by the superzombies, and most of its members are killed; L.J. eventually succumbs to the T-virus and bites Carlos before being shot dead, Mikey was devoured by a band of super-zombies, and Chase falls from his sniper position in the Paris Las Vegas Eiffel Tower replica while fighting with the super-zombies who managed to locate his position. Dr. Isaacs tries to control Alice via satellite, but she is powerful enough to hack Umbrella's computer network and find out that Isaacs is just a few meters away on the roof of a nearby building. She attacks the few Umbrella men and kills all of them but Isaacs, who manages to escape by helicopter after being bitten by his own creatures.

The few survivors decide to raid a nearby Umbrella facility, hoping to use their helicopter to fly to Alaska. Inside the underground laboratories, Isaacs tries to save himself by injecting more and more antivirus, but he seems helpless and begins to exhibit signs of mutation as a result of the overdose. His second-in-command, Captain Slater, was sent by Wesker to kill him due to his failure and insubordination, but Isaacs proves resistant to gunfire and kills Slater and some nearby guards with his new tentacled hand. He then begins to kill everyone in the base, but he's trapped inside the lower levels by the White Queen, the computer which controls the facility.

The convoy arrives to the facility, only to find out that its perimeter is surrounded by thousands of undead. Carlos, believing his infection to be beyond any cure, chooses to sacrifice himself in order to open a safe path for the rest of the convoy to pass through. He gets into a gasoline truck with some dynamite, drives into the crowd and detonates the explosives. Claire, K-Mart, Liam and the remaining surviving convoy members gets into the chopper and flies away to Alaska, leaving the convoy's fate open to viewers. Alice, meanwhile, decides to get her revenge on Umbrella.

When she gets into the labs she is told by the White Queen, who confirms that the real Alice's blood is the ultimate cure for purging the world of the biohazard, that she has to reach to lower level in order to create a cure. Down there, she finds one of her clones, but before she can examine it, Isaacs now a Tyrant, attacks her. She stabs him with a knife and he flees and is able to instantaneously heal his stab wound. The clone wakes up, seemingly dying in Alice's arms. A battle of telekinetic powers then continues inside the Mansion, and in the replica of the laser corridor from the Hive. Isaacs taunts Alice, saying that he is the "future", with Alice retorting to Isaacs they are both going to die, and the laser cuts him into pieces, but stops just before reaching Alice. Her clone is revealed to have been the one to turn it off.

In Tokyo, Wesker informs the rest of Umbrella that contact with the North American branch has been lost, and decided to control further experimentation from his Japan facility. Using their holographic technology, Alice joins the meeting and informs them that she is after them and "bringing a few of her friends". The film ends as Alice, next to one of her clones, watches hundreds of others awaking.