In the distant past, the spacecraft of an advanced humanoid alien race arrives on Earth. One of the aliens consumes a dark liquid, causing its body to disintegrate and fall into a nearby waterfall - seeding Earth with the building blocks of life.
In 2089, archaeologist couple Elizabeth Shaw and Charlie Holloway discover a star map among the remnants of several otherwise unconnected cultures. They interpret this as evidence of humanity's forerunners. Peter Weyland, the elderly founder of the Weyland Corporation, funds the creation of the scientific vessel Prometheus to follow the map to the distant moon LV-223. The ship's crew travels in stasis while the android David monitors their voyage. In 2093, the ship arrives, and its crew are informed of their mission to find the ancient aliens, called "Engineers". Mission director Meredith Vickers orders them to avoid any direct contact and return if the aliens are found. The Prometheus lands near an alien temple. A team including Shaw, Holloway and David explores the temple, while Vickers and Captain Janek monitor their progress onboard the ship.
They find several ampoule-like artifacts, a monolithic statue of a humanoid head, and the corpse of a giant alien, presumed to be one of the Engineers. Other bodies are later found and the species is presumed to be extinct. David secretly returns an ampoule to the ship, while the remaining ampoules begin leaking dark liquid. An approaching storm cuts the expedition short, and the crew is forced to return to Prometheus. Botanist Milburn and geologist Fifield are separated from the expedition and stranded in the temple. In the ship, Shaw and medic Ford analyze the Engineer's head, and discover that its DNA is almost identical to that of the human species. Meanwhile, David investigates the ampoule and discovers a dark liquid substance. He intentionally infects Holloway with the substance. Later, Shaw and the infected Holloway have sex.
In the temple, Fifield and Milburn are attacked by snake-like creatures. Milburn is killed, and a corrosive fluid from one of the creatures melts Fifield's helmet, exposing him to the dark liquid leaking from the ampoules. The crew return to the temple and find Milburn's corpse. David discovers an uncharted room containing a living Engineer in stasis and a star map highlighting Earth. Elsewhere, Holloway's infection rapidly ravages his body, and he is rushed back to the ship. Vickers refuses to let him onboard, and burns him to death at his own request. A medical scan reveals Shaw is pregnant, despite being sterile. A desperate Shaw uses an automated surgery table to cut a squid-like creature from her abdomen. Weyland is found to have been in stasis aboard the ship, and explains to Shaw that he intends to ask the Engineers to help him avoid his impending death.
A mutated Fifield attacks the hangar bay and kills several crew members before being killed himself. Janek suggests the planet was used by the Engineers as a military base until they lost control of their biological weapons; the ampoule. The remaining crew return to the temple and awaken the Engineer. David explains their situation to the Engineer, who responds by decapitating David and killing Weyland and Ford. Shaw escapes the alien ship as it is activated by the Engineer. The still-active David reveals it is going to Earth to unleash the ampoule on humanity. Vickers orders Janek to return to Earth, but Shaw convinces them to stop the Engineer ship by any means necessary. Janek crashes the Prometheus into the Engineer ship while Vickers uses an escape pod. The disabled Engineer ship crashes to the planet, killing Vickers. Shaw goes to the escape pod to retrieve supplies. Inside, she finds her squid-like offspring, which has grown to gigantic size. The Engineer survives the crash and attacks Shaw, who releases the tentacled monster. It subdues the Engineer by thrusting a tentacle down its throat. Shaw recovers David's remains from the alien ship, and together they activate another Engineer ship to travel to the Engineer homeworld in an attempt to understand why they created humanity and attempted to destroy it.