Final Fantasy VII: Dirge of Cerebrus's main character is Vincent Valentine. His story and his past are both explained in the game, as well as his origins.

Reeve Tuesti, the former head of Shinra's Urban Development Department, leads the World Regenesis Organization (WRO; called World Restoration Organization in the Japanese version of the game), an operation dedicated to restoring the world and undoing the mistakes made by Shinra. Prominent members of WRO include Cait Sith, assisting both Reeve and Vincent in certain parts of the game; Yuffie Kisaragi, in charge of espionage and intelligence gathering; Shalua Rui, WRO's chief scientist; and Cid Highwind, head of the WRO's airship division. Cloud Strife, Barret Wallace, and Tifa Lockhart also lend their support to the WRO during the second half of the game.

The Tsviets are the main antagonistic forces in Dirge of Cerberus: Final Fantasy VII. The Tsviets consist of the following members: Azul the Cerulian, Shelke the Transparent, Rosso the Crimson, Nero the Sable, and Weiss the Immaculate. Vincent fights these enigmatic forces during the course of the game.

Other characters that were featured prominently or otherwise in Final Fantasy VII also return. Lucrecia Crescent is shown mainly in flashbacks, further fleshing out her story. Hojo returns as well in flashbacks, and he then reappears in the penultimate moments of the game. Red XIII also makes a brief cameo in the game's ending, though he does not speak.

Grimoire Valentine, Vincent Valentine's father, is introduced in Dirge of Cerberus. He appears in the game's flashbacks with Lucrecia Crescent, serving as her mentor.

Omega WEAPON appears as the game's final boss and is the result of Professor Hojo's plans to merge with Weiss and bring destruction upon the earth.

A new character, Genesis, appears in the secret ending of the game. An enigmatic character, the Deep Ground soldiers and the Tsviets are the result of his evolution.

Usher and Argento are two characters who are featured only in the Japanese version of the game, specifically in the multiplayer mode; Usher gives assistance to the player many times throughout and Argento serves as an instructor. The Restrictors are also a set of characters that appear in the Multiplayer mode; they serve as the commanding force of Deep Ground, before they are succeeded by the Tsviets.