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- Fodder — These feral bipeds roam the craft in search of food. They are the weakest of the enemies faced on the Sphere and can be killed easily.
- Harvester — Beings who apparently have a vital role in harvesting the protein from captives to feed the Sphere. They come in two varieties, one which walks around on mechanical legs (which can be blown off) and can travel freely through a network of organic tunnels, and one which has attached an antigravity device to itself, allowing it to fly through the air freely. This version has been said to bear resemblance to the Assault Commanders from Duke Nukem 3D. Both kinds of Harvesters are armed with Crawler Launchers, but only the walking variety use the alternate fire.
- Hunter — The foot-soldiers of the invading force, it is their responsibility to track down and eliminate any captives that get loose. They are armed with the standard Rifle, and will use both firing modes, depending on how far away they are from Tommy. The majority of the enemies faced in Prey are Hunters.
- Mutilated Human — These unfortunates are humans who have had painful-looking machinery implanted into their bodies, leaving them in a zombie-like state, fit only to perform menial, repetitive tasks for the Sphere. They only fight when provoked (by either shooting at them or getting too close) and will continue to fight even after being decapitated.
- Hound — Despite the name, Hounds are as large as lions and serve as the side kicks to Hunters. When attacking, they can knock Tommy around and temporarily impair his vision. They can also rear up on their hind legs and walk on them for better striking. They resemble the Xenomorphs from the Alien films.
- Gasbag — Floating, tentacled creatures which spit acid. Gasbags will attack anything that moves near them, whether it is Tommy or a Hunter.
- Mutate — Born of the melding of the DNA of human and alien victims of the living Sphere. The Mutate is a surprisingly nimble opponent despite its bulk, and possesses a massive claw that it uses to impale its enemies in a spinning attack. But they have poor lateral agility so it is very easy to dodge them when they charge.
- Drone — Drones are flying robots that maintain and repair the mechanical parts of the Sphere. They're also equipped with some ranged weapons to repel intruders.
- Wraiths of Fallen Spirits — When Tommy loses all his health, he enters the Death Realm. He must battle the Wraiths of Fallen Spirits to regain both health and spirit energy before returning to his living body. These dishonored spirits inhabiting the realm of the spirit world also seem to inhabit the sphere. This is probably because their souls are bound to the sphere because of their dishonored deaths at the hands of the Sphere. One of the examples of these wraiths is in a scene where it enters a holding chamber and possesses a child who abruptly is used to brutally murder the other child in the cell. This is an indication that these wraiths are similar to, if not the same as, demons in other religions, who possess hosts to perform terrible acts of evil. A short time later, possessed children attack on two occasions, including one scene where a school bus is encountered. However, these children only appear relatively early in the game and are conspicuously absent for the remainder. The human possession aspect is neither further explored nor resolved, leaving something of a loose end in the plot. However, the Wraiths occasionally appear at various points and fly around, sapping spiritual energy if they come in contact. It's interesting to note that the Sphere Mother, after first seeing a wraith, states that they were not on the sphere until Tommy's return from his first visit in the spirit realm. Rather than being upset, she instead finds it intriguing.