USS Delta team enters the Raccoon City Underground Laboratory, where they meet up with Alpha team leader HUNK. Their mission is to assist Alpha team in stopping Dr. Birkin from handing over his T-Virus research to the U.S. military and retrieve the G-Virus. On their way to Birkin's lab, they find Birkin has paid numerous UBCS mercenaries to work for him while the deal goes forward. When they reach Birkin's lab, the doctor is shot, and HUNK and another Alpha leave with the samples. They soon find that Birkin survived the attack and infected himself with the virus. The Birkin creature proceeds to kill off most of Alpha team before disappearing; HUNK offers to go back in search of the sample.
Not long after the battle, it becomes evident that the T-Virus has leaked city-wide, and people are beginning to transform into flesh-hungry zombies. In what they see as a punishment by USS Command, Delta team is ordered to remove evidence of Umbrella's role in the outbreak. Heading into Raccoon City Hall, Delta team meets with a UBCS mercenary and they monitor Nicholai Ginovaef; he is soon revealed as a traitor and attempts to kill Delta team.
Later, the team is sent out around Raccoon Park to find the Nemesis-T Type, which has gone rogue. A second parasite is injected into its body in order to bring it back under control. Shortly after this mission is completed, the team is then sent out to the Raccoon City Police Department, ordered to kill any surviving police officers and destroy evidence linking the company to the outbreak. When this is done, the team exits the station, and soon after encounter Leon S. Kennedy, whom they begin to hunt down along with Sherry Birkin. After they find and corner Leon, Claire Redfield and Sherry, the game can end in two ways: in one ending, the team resigns from Umbrella over their abandonment during the mission and betrays them by letting the three live; in the other, Leon and Claire are executed and Sherry is sent to an Umbrella facility.
There are twelve playable characters in the game, with six characters for each side. On the Umbrella Security Service team there is: Vector, the team's recon expert and is equipped with a cloaking ability; Beltway, who is proficient in the use of explosives; Bertha, the medic; Spectre, the marksman; Four Eyes, the scientist, with the ability to program the bio-organic weapons (BOWs); and Lupo, who is the team leader. On the United States Special Ops team there is: Willow is the recon expert; Dee-Ay is the team leader; Tweed is the demolitions expert; Harley is the medic; Shona is the field scientist; and Party Girl is the sniper.
Alongside the single player mode, the game also offers four-player co-operative Raccoon Mode, which pits the USS against the U.S. Special Operations Teams.
The zombies in the game are able to attack the player in a multitude of ways. A zombie that gets too close to the player will usually attempt to grapple the player, prompting the player to rapidly shake his/her analogue stick to avoid getting bitten. Players who are unsuccessful in doing this will be infected for a limited amount of time. Whilst they are infected they gain small bonuses at the expense of a slowly depleting health bar. If a player is successfully bitten and later runs out of time after becoming infected, the player will lose control of their character and begin attacking their former teammates until killed, allowing the player to respawn. There will be additional enemy types in the game besides zombies, such as Lickers, Hunters, and Cerberuses. Furthermore, the player will be able to control them under certain circumstances.
Heroes Mode is an online multiplayer feature allowing to play characters such as Leon S. Kennedy, Claire Redfield, Jill Valentine and Carlos Oliveira, plus Ada Wong, HUNK, Nicholai Ginovaef and a new character, called Lone Wolf. Xbox 360-exclusive Nemesis Mode allows one player to control Nemesis and use him to kill the other team.