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- Amanda Ripley is the main playable character in the game. She is drawn to Sevastopol Station by the chance to discover what happened to her mother Ellen when the USCSS Nostromo disappeared fifteen years previously.
- An unnamed Drone is loose aboard the station and is responsible for the slaughtering of numerous residents, as well as establishing a Hive in its engineering levels.
- Christopher Samuels is a synthetic working for the Weyland-Yutani Corporation who organises the expedition to Sevastopol to recover the Nostromo's flight recorder. When things go disastrously wrong aboard the station, Samuels helps the human survivors to control the situation.
- Marshal Waits is the commanding officer of the Colonial Marshal Bureau division stationed aboard Sevastopol. He comes up with a plan to destroy the Drone by trapping it in a remote area of the station and jettisoning the module into space.
- Deputy Ricardo is one of Waits' deputies. He assists Ripley in dealing with the catastrophe on Sevastopol, directing her around the station as she attempts to contain the situation. He is also the playable character in The Trigger DLC.
- Diane Verlaine is captain of the Torrens, the ship that brings Ripley and the others to Sevastopol. She and her ship stay a safe distance from Sevastopol as the crisis unfolds, but later docks with the station in an attempt to rescue Ripley.
- Nina Taylor, a Weyland-Yutani lawyer who accompanies the mission to Sevastopol. Unknown to the others, she is fact on a secret mission for the company seeking to recover the Xenomorph and return it for study.
- Henry Marlow is the captain of the Anesidora, the ship that unwittingly brought the Drone to Sevastopol. After recovering the Nostromo's black box from deep space, Marlow elects to follow the flight path in the hope of salvaging the ship. Instead he finds the Xenomorph Eggs aboard the derelict on LV-426. The player briefly plays as Marlow in a flashback to the events on LV-426.
- Axel Fielding is a surviving inhabitant of Sevastopol, and one of the first people Ripley encounters after boarding the station. Although initially hostile, he agrees to assist her in exchange for escape aboard the Torrens. He is also the playable character in the Lost Contact DLC.
- Dr. Kuhlman is one of the three doctors who were stationed in the San Cristobal Medical Facility aboard the station. He aids Ripley in recovering medical supplies after Taylor is wounded during a spacewalk.
- B. Ransome is a senior executive aboard Sevastopol, hoping to benefit himself financially from the Xenomorph that is slaughtering its inhabitants. Although he is never seen by the player, various reports and messages from him can be found on Sevastolink terminals in the game. He is also the playable character in the Corporate Lockdown DLC.
- Dr. K. Lingard is one of the three doctors who were stationed in the San Cristobal Medical Facility aboard the station. She is also the playable character in the Trauma DLC.
- F. Sinclair is a survivor aboard Sevastopol. Originally the leader for Seegson Security, he later became the leader of a violent and dangerous group of survivors after convincing the security force to disband from their positions and form a group together for greater chances of survival.
- Mike Tanaka is a survivor aboard Sevastopol. He sketches multiple pictures of residents aboard the station as well as some of the Drone. He is killed before Ripley arrives on the station.
- Catherine Foster is a crewmember aboard the Anesidora, and Captain Marlow's wife. Foster was the one who was impregnated by a Facehugger from the derelict on LV-426, and as a result, she unknowingly brought the Drone aboard Sevastopol.
- Hughes is a survivor aboard Sevastopol. He is seen by Ripley, getting violently killed by a Working Joe.
- Heyst is a crewmember aboard the Anesidora.
- Meeks is a crewmember aboard the Anesidora.
- Lewis is a crewmember aboard the Anesidora.
- William Connor is the navigator and communications officer aboard the Torrens. He stays on the ship with Verlaine during the crisis on Sevastopol.
- A second Drone gets aboard the Torrens, possibly killing its crew, before cornering Ripley upon her return to the vessel.
- Julia Jones is a reporter who travels to Sevastopol to report of the decommissioning of the station. Instead, she finds herself in the middle of the Xenomorph horror. Her reports can be found throughout the game on Sevastolink terminals.
- W. "Chief" Porter is a survivor aboard Sevastopol, and the chief engineer. He is responsible for the construction of the Bolt gun.
- G. Spedding is the android liaison executive for Seegson Synthetics.
- Smythe is head of synthetic development for Seegson Synthetics.
- Suzanne Archer is a receptionist for Seegson Synthetics. She was in a relationship with Spedding.
- D. Turner is a fellow marshal like Waits, who is killed before Ripley arrives.
- E. Winters is a survivor aboard Sevastopol. She keeps note of her experiences as a survivor in a few audio logs.
- Zachary Watson is an alcoholic resident aboard Sevastopol who is killed before Ripley arrives.