- Elizabeth Shaw: A young scientist who is part of the crew of the spaceship Prometheus. She is driven by her faith to find the Engineers, the creators of humanity.
- David: An android who is also a member of the Prometheus crew. He is highly intelligent and curious, but also ruthless and calculating.
- Weyland: The CEO of Weyland Corp., the company that funded the Prometheus expedition. He is obsessed with finding the Engineers and gaining immortality.
- Meredith Vickers: The Weyland Corp. representative on the Prometheus. She is a ruthless and ambitious businesswoman who is willing to do anything to achieve her goals.
- Janek: The captain of the Prometheus. He is a veteran astronaut who is trying to keep his crew safe while also fulfilling the mission objectives.
- Holloway: A biologist on the Prometheus who is Shaw's romantic partner. He is fascinated by the Engineers and their creations.
- Fifield: A geologist on the Prometheus who is eager to explore the alien moon. He is reckless and impulsive, often putting himself and his crewmates in danger.