The tarantula hawk wasp is the biggest natural enemy of the tarantula. Many animals and insects, including larger mammals, reptiles, birds and fly maggots, eat tarantulas as well.

The tarantula hawk wasp hunts for the tarantula, finds its burrow and lures it out. The wasp then stings the tarantula, paralyzing it. Afterwards, the wasp drags the tarantula to its lair and lays an egg on the spider. When the wasp maggot hatches, it eats the tarantula. Larger mammals, such as coyotes, foxes, weasels and skunks, have been known to eat tarantulas. However, they often given up shortly after starting due to the spider's barbed hairs and agile movements. Reptiles, such as large lizards and snakes, sometimes eat tarantulas. However, this can prove dangerous as tarantulas have also been known to eat lizards and snakes. Birds, such as hawks, eagles and owls, sometimes eat tarantulas as well.

Flies sometimes eat tarantulas in a fashion similar to the tarantula hawk wasp. The fly lays its eggs on the tarantula, and when the eggs hatch, they burrow into the spider and eat it alive from the inside out. As the eggs turn into flies, they emerge, bursting the spider's abdomen and killing it. Tiny mites also feed on tarantulas but don't typically cause death.