Los Ganados can dodge, wield melee and projectile weapons, and are capable of working collectively and communicating with each other.

The main enemies are violent villagers referred to as Los Ganados ('The Cattle' in Spanish). These are often incorporated into the game's many boss fights, in which the player must avoid instant-kill attacks. There are also quick time events, in which the player must press buttons indicated on-screen to execute actions such as dodging a falling boulder or wrestling an enemy to stay alive. Based on the situation, players can interact with the environment: kicking down a ladder, jumping out of a window, dodging an attack or executing a 'finishing move' on weakened enemies. Resident Evil 4 adds context-sensitive controls.

The laser sight enables the player to target key hit zones on enemies. Unlike previous entries in the series, Resident Evil 4 has the camera following directly behind the main character.