The main gameplay feature of Resident Evil Zero, dubbed the "partner zapping" system, is unique in the series in that instead of choosing a single character to play through the whole game, the player controls the two main characters, Rebecca Chambers (S.T.A.R.S. Bravo Team's 18 year old medic, seen in the original Resident Evil) and Billy Coen (an escaped convict) simultaneously through the entire game. Both characters can travel together, with one character being controlled by the player and other by the computer, or split up and search separate areas each at the same time. This dual control is central to solving some of the puzzles in the game. The difference between both characters is that Rebecca can acquire chemicals with her mixing kit and can get into tight spaces, but is more fragile to enemy attacks. In contrast, Billy can handle heavy objects, has an oil lighter and can sustain more damage than Rebecca, but cannot mix herbs (in contrast from previous games, in which any character could mix herbs).

The game also does away with the use of boxes placed in fixed locations for the player to store items in. Instead, the player can now drop items on the floor to make room for new ones, which they can later retrieve for future use. The locations of dropped items are displayed on the player's map

On July 23, 1998, S.T.A.R.S. Bravo Team is sent in to investigate a series of grisly murders in the Arklay Mountains region outside of Raccoon City. On the way to the scene, Bravo's helicopter malfunctions and is forced to crash land in the forest. Luckily, there are no fatalities. The team soon discovers an overturned Military Police transport truck, along with the mutilated corpses of two officers. The team soon splits up and Bravo team's field medic, rookie member Rebecca Chambers, stumbles upon a train suspiciously stopped in the middle of the forest- the Ecliptic Express. Soon thereafter, she discovers that it's infested with zombies. Rebecca begins to wonder what she has gotten herself into. After a little exploration, she teams up with the man the Military Police were escorting to his execution; former Marine Billy Coen, and the two of them face off against a band of zombies, swarms of strange leeches, and a mysterious individual with possible links to the disaster. As with previous installments, the two characters visit various locales through the course of the story, including a train (aforementioned Ecliptic Express), a research facility, a laboratory and a factory. The Umbrella's underground facility from Resident Evil 2 is briefly visited by Rebecca at one point of the game as well.

Other recurring Resident Evil characters present are Alpha Team leader Albert Wesker and Umbrella scientist Dr. William Birkin. The two make appearances in a few cutscenes, but never interact with the main characters. The game also introduces Dr. James Marcus, one of the three founding members of Umbrella and the true creator of the T-Virus.