The film begins immediately following the events of Alien vs. Predator, onboard the Predator spaceship leaving Earth which is carrying dead Aliens, living facehuggers, and the body of the dead Predator who had defeated the Alien queen. A chestburster erupts from the dead Predator's body; it is a new creature which is a hybrid of Alien and Predator characteristics (referred to as the "Predalien" by the film’s production team). It quickly grows into an adult and begins killing the Predators onboard the ship. One Predator fires at it, creating a hole in the hull and causing the ship to crash in the forest outside of Gunnison, Colorado. With the Predators dead, the Predalien and several facehuggers escape into the forest and impregnate a father and son on a hunting trip as well as several homeless people living in the sewers.
A distress signal from the ship reaches the Predator homeworld. A lone Predator responds to the signal and comes to Earth, using his advanced technology to observe the cause of the crash and track the facehuggers. He begins to erase the evidence of the Aliens' presence, first by blowing up the crashed ship and then by using a blue liquid to dissolve the bodies of the facehuggers and their victims.
Meanwhile, in Gunnison, ex-convict Dallas Howard has just returned to town after serving time in prison. He is greeted by Sheriff Eddie Morales and reunites with his younger brother Ricky. Ricky has a romantic interest in the more affluent Jesse and is being harassed by her current boyfriend Dale and his cohorts. Dale throws Ricky's keys into the sewer drain, and while Dallas and Ricky search for them they hear noises and see evidence that something strange is in the sewers. Meanwhile, Kelly O'Brien has also just returned to Gunnison from deployment in the army and is reunited with her husband Tim and daughter Molly. Sheriff Morales leads a search party into the forest to search for the father and son who have gone missing on their hunting trip. A deputy encounters and is killed by the Predator.
The Predator makes his way into the sewers and fights several adult Aliens. The battle reaches the surface, where the Aliens disperse into the town. The Predator pursues several of them to the city’s power plant, where the explosions caused by his weapons fire cause a citywide blackout. Ricky and Jesse meet at the high school swimming pool, but are interrupted by Dale and his friends just as the blackout occurs and an Alien enters the building, killing Dale’s friends. An Alien also invades the O’Brien home, killing Tim while Kelly escapes with Molly. They meet up with Ricky, Jesse, Dale, Dallas, and Sheriff Morales at a sporting goods store to gather guns. National Guard troops arrive but are quickly killed by the Aliens. The battle between the Predator and the Aliens enters the sporting goods store, where Dale is killed and the Predator’s shoulder cannons are damaged. He is able to modify one into a handheld blaster.
As the survivors attempt to escape the town they make radio contact with Colonel Stevens, who indicates that an air evacuation is being staged at the center of town. Kelly, however, is suspicious of the military’s intentions. While Sheriff Morales heads to the evacuation area with the rest of the surviving townsfolk, the remainder of the group opts to head for the hospital, where they may escape via helicopter. The hospital, however, has been invaded by the Aliens and the Predalien who have set up a hive there. The Predalien demonstrates the ability to implant embryos directly into host humans without the need for eggs or facehuggers. The Predator also arrives at the hospital, and in the resulting battle Jesse is killed, Ricky is injured, and Dallas takes possession of the Predator’s blaster cannon.
As the battle reaches the rooftop Dallas, Ricky, Kelly, and Molly escape in the helicopter while the Predator and Predalien battle hand-to-hand. The two creatures mortally wound each other just as the military jet arrives and is revealed not to be a rescue airlift, but rather a tactical nuclear strike. The Predator, Aliens, Predalien, and remaining citizens are eradicated in the blast along with the rest of the city. The blast causes the helicopter to crash in a clearing, where the survivors are rescued by the military and the Predator’s blaster cannon is confiscated.
In the closing scene, Colonel Stevens presents the blaster cannon to a Ms. Yutani (foreshadowing the Weyland-Yutani corporation of the Alien series). She remarks that the world is not ready for this technology, to which he replies: "But this isn’t for our world. Is it, Ms. Yutani?"
* Reiko Aylesworth as Kelly O'Brien. Having just returned to Gunnison following a tour of duty in the military, Kelly reunites with her husband Tim and daughter Molly.
* Ariel Gade as Molly O'Brien, the daughter of Kelly and Tim. Having not seen her mother for some time due to her military service, Molly is somewhat estranged from her and does not immediately adjust to her return.
* Kristen Hager as Jesse, the attractive classmate of Ricky. She is unhappy with her current boyfriend Dale and begins to flirt with Ricky, noting his affections towards her.
* Robert Joy as Colonel Stevens, the commander of the military forces attempting to contain the Alien infestation. He lures the people of Gunnison to the center of town with the promise of an airlift rescue, but his true intention is to destroy the town with a tactical nuclear strike in order to eliminate the Aliens.
* Johnny Lewis as Ricky Howard, the younger brother of Dallas. Ricky is a high school student with a job as a pizza delivery boy. He is romatically attracted to the more affluent Jesse but is bullied by her current boyfriend Dale and his cohorts.
* John Ortiz as Sheriff Eddie Morales. As the sheriff of Gunnison, Eddie leads a search party to look for the missing townsfolk in the forest and attempts to contact the military and evacuate the citizens during the Alien infestation.
* David Paetkau as Dale Collins, the boyfriend of Jesse. He bullies Ricky and challenges him when Jesse shows an interest in Ricky.
* Steven Pasquale as Dallas Howard. Dallas has just returned to Gunnison after being released from prison. He shares a friendship with Sherrif Morales and reunites with his younger brother Ricky.
* Sam Trammell as Tim O'Brien, the husband of Kelly and father of Molly.
* Ian Whyte as the Predator, one of the film's titular alien species. The main Predator of the film comes to Earth in order to eliminate the Aliens and all traces of their presence. Whyte had previously portrayed the Predator in Alien vs. Predator. Additional Predators in the film's opening scene were played by Ian Feuer and Bobby "Slim" Jones.
* Tom Woodruff, Jr. as the Aliens, the other titular species of the film. Having previously portrayed the Aliens in Alien 3, Alien Resurrection, and Alien vs. Predator, Woodruff reprised the role for this film. He also portrayed the Alien/Predator hybrid creature, dubbed the Predalien by the production team.