The game's main protagonist is Leon S. Kennedy, one of the few survivors of the Raccoon City incident, who was recruited and trained by the US Government to become a special agent after they learned of his actions in Raccoon City. Now an agent for a special security agency, Leon is sent to Spain on a mission to rescue Ashley Graham, the President's daughter, who has been kidnapped by a mysterious cult organization.
Leon travels to an undisclosed village in Spain where he encounters a horde of unruly villagers who pledge their lives to Los Iluminados, the cult that perpetuated Ashley’s kidnapping. He meets Luis Sera, a former Los Iluminados researcher, who aids Leon on his mission. By examining Sera’s notes, Leon discovers that Los Iluminados gained control of their subjects by implanting a mind controlling parasite known as "Las Plagas" into their bodies. Unfortunately, Leon and Luis are captured, during which time Leon is injected with the parasite. After escaping and meeting a mysterious man known as the Merchant, who supplies Leon with ordnance and weaponry, Leon returns to his objective. He travels across a lake where he faces a large and dangerous creature, "Del Lago". Leon defeats the creature, but is severely fatigued, and collapses in a cabin. After regaining consciousness, he obtains a key to open the church where Ashley is being held. On the way back Leon fights with the gigantic "El Gigante". While escaping with Ashley, he meets the mastermind of Los Iluminados: Osmund Saddler. He tells them about the Plagas growing in their bodies. The two escape from Saddler, and make their way to the extraction point. Meeting up with Luis once more in a cabin, they are ambushed by a seemingly never-ending crowd of Ganado. When the attack ends, Leon and Luis separate. However, Leon finds out that the helicopter has been shot down. After traveling through a canyon, where the two face another El Gigante. Traveling down a gondola, and facing village chief Bitores Mendez, Leon takes his false eye, and uses it on a retinal scan to open a door, which leads to the castle. Leon and Ashley believe they can take refuge in it until another chopper arrives. They were wrong.
After meeting yet another of Saddler's acolytes, the castle's castellan, Ramon Salazar, who jacks Leon's communication with Washington, Leon and Ashley travel to the center of the castle. But Ashley starts coughing blood, which distresses her, and she runs off, and is captured. While searching for Ashley, Leon meets Ada Wong, who survived the destruction of the Umbrella underground lab and her injuries from the tyrant at the lab's self-destruct mechanism is there for the sample of the "Dominant Species Plaga" for her enigmatic employers, the 3rd Organization, but she doesn't tell Leon about it. Then Leon fights his way through the underground sewer system, despite assaults by Saddler's mutated "Novistadors". When Leon finds Ashley and is about to rescue her, Luis runs in with drugs to suppress the growth of the parasite, and a sample of Dominant Species Plaga for the U.S. government, when he is killed by Saddler. Saddler retrieves the sample and leaves. Leon rescues Ashley, but vows to avenge Luis' death. While Leon can't get to Ashley, she must find a way to him. She is, however, caught again and sent to the military research island, forcing Leon to travel to there too with Ada after defeating Ramon Salazar. After numerous altercations with Saddler's fiends (including his former ally Jack Krauser and It), Leon is able to successfully rescue Ashley, and run from Saddler with Ada's assistance.
After an intense fight with Saddler, Leon kills him with a special Rocket Launcher. He finds the Sample but Ada forces him to give it to her. She then escapes from the complex on a helicopter, leaving Leon and Ashley to escape via jet-ski. Although Leon and Ashley manage to escape from the island, the sample's whereabouts and fate remain uncertain.
Ada arrives in the village seemingly around the same time as Leon. After fighting off a pair of Ganados, Ada is contacted by Wesker and is told to ring the church bell to pacify the villagers. She then notices Leon. having been ambushed by a gang of villagers. Ada then heads to the church while avoiding contact with Leon and effectively ringing the bell, which in turn saves Leon for the Ganado's assault.
When we next see Ada, she is searching through a book about the villagers in the home of Bitores Mendez when she gets another call from Wesker. He informs her that their infomant of sorts, Luis Sera, has been captured by the Ganados and is being held in a cabin. Since he is needed to gain access to the Plaga sample Ada is sent off to rescue him. Ada arrives just in time to see Luis and Leon being moved to a new location. On her way back to Bitores' house she witnesses Leon being assaulted by Bitores and intervenes, making him chase her instead of harming Leon further. She makes her way down the back from the house and is tranquilized by a Ganado accompaning Bitores' party chasing her. She wakes moments before meeting her end by an axe. She then meets with Luis after the cabin assault and he informs her he is off to gather the sample.
Ada then reveals herself to Leon within the castle, then after a short conversation, escapes to the hedge maze outside and receives another message from Wesker telling her to get the sample from Luis, and if she encounters him, to eliminate Leon. While doing her best to avoid Leon, she meets up with Luis just in time to see Saddler kill him and take back the sample
Ada and Jack Krauser then meet in the island's radio tower. Wesker then contacts her and informs her that Krauser is now under orders to assassinate Leon. Ada makes her way to where the two are fighting and shoots the knife out of Krauser's hands, that was mere inched from his chest. After Leon's fight with Krauser, Ada reports Krauser's death to Wesker. She then meets up with Leon who, being temporarily under his Plaga's control, attacks Ada. Soon after she is attacked by none other than a mutated Krauser, who had not fallen in battle to Leon. Not wanting to deal with having reporting a living man dead to Wesker, she finishes of Krauser once and for all.
Ada then arrives through a balcony enterance in Ashley 's containment room, and distracts Saddler while Leon and Ashley escape. Thinking she defeated Saddler, Ada attempts to retrieve the Plaga sample, only to be sneak attacked and captured by Saddler. Being released from captivity by Leon, Ada races to obtain a rocket launcher to assist the struggling Leon in battle(mirroring her assistance to Leon back in Resident Evil 2 with a similar concept). After the battle Ada steals the Las Plaga sample Leon had gained from the defeated Saddler. She then makes her escape from the island via a helicopter and gives Leon keys to a jet-ski to make his escape with Ashley as Ada sets the island to explode. In the last scene of her report, Ada states how the organization she is working for: wants the Plaga sample for themselves, that she was ordered to pretend to work with Wesker which she found "entertaining" and that she intends to send Wesker a different sample as ordered. She then states that "Wesker is not stupid, that he know exactly what the Organization is planning, and that the mission may be over but the battle is just beginning"