In the underground Facility, senior technicians Gary Sitterson (Richard Jenkins) and Steve Hadley (Bradley Whitford) discuss plans for a mysterious ritual. A similar operation undertaken by their counterparts in Stockholm has just ended in failure.
American college students Dana Polk (Kristen Connolly), Holden McCrea (Jesse Williams), Marty Mikalski (Fran Kranz), Jules Louden (Anna Hutchison), and Curt Vaughan (Chris Hemsworth) are spending their weekend at a seemingly deserted cabin in the forest. From the facility, where they possess some technological control over the cabin, Sitterson and Hadley manipulate them by intoxicating the teenagers with mind-altering drugs that hinder rational thinking and increase libido. They take bets from the different facility departments on what kind of monster will attack the teenagers and discuss the failures of similar rituals in other nations.
In the cabin's cellar, the group finds many bizarre objects, including the diary of Patience Buckner, a cabin resident abused by her sadistic family. Dana recites incantations from the journal, inadvertently summoning the zombified Buckner family. By releasing pheromones, Hadley successfully induces Curt and Jules to have sex. Attacked by marauding Buckners, Jules is decapitated, but Curt escapes, alerting everyone else. Marty, a frequent marijuana smoker, discovers concealed surveillance equipment before being dragged off by a Buckner. Later, the Facility workers learn that the ritual in Japan has also ended in failure, ending Japan's streak of success, and confirming that the American ritual is humanity's last hope.
Curt, Holden, and Dana attempt to escape in their RV, but Sitterson triggers a tunnel collapse to hinder their flight. Curt jumps a ravine on his motorcycle, only to crash into a camouflaged force shield, killing him. Holden and Dana retreat to the RV to re-evaluate, but one of the Buckners is waiting within and stabs Holden as they are driving away, resulting in the RV veering into a lake. Dana swims ashore and is beset in turn. As she is attacked, the staff celebrate her impending death and the successful completion of their ritual.
The celebration is interrupted by a phone call from "downstairs" pointing out that Marty has survived. He rescues Dana and shows her to a hidden elevator. They take the elevator into the Facility, where a menagerie of monsters are imprisoned. Dana correlates them with the knickknacks in the cabin's basement and realizes that those items dictate which monster will be unleashed. Cornered by the Facility's security troops, she and Marty release the multitude of monsters, including zombies, a werewolf, a giant snake, wraiths, a unicorn, and a merman, among others, who wreak havoc and slaughter the staff.
Fleeing further, Dana and Marty discover a temple where they are confronted by the project's Director. She explains that, every year, worldwide rituals are held to appease the Ancient Ones; malevolent beings living beneath the surface of the earth, one of them under their own Facility. The Ancient Ones are kept in perpetual slumber through an annual pars pro toto sacrifice of five young people embodying certain archetypes: the whore (Jules), the athlete (Curt), the scholar (Holden), the fool (Marty), and the virgin (Dana). The order in which intended victims perish is flexible, so long as the Whore dies first and the Virgin survives or dies last. The Director urges Dana to shoot Marty, completing the ritual and saving humanity. But the standoff is interrupted by a Werewolf that attacks Dana. Patience Buckner suddenly appears and kills the Director. Concluding that humanity is not worth saving at the expense of innocent lives, Dana and Marty share a joint as an Ancient One stirs, its giant hand emerging from beneath the temple floor, destroying the cabin and the Facility, ending the film.