Enemies are one of the main obstacles found in Super Meat Boy. Enemies, as well as horrible death traps and salt, can kill Meat Boy on contact. The first enemies of the game are Dust Bunnies and Betus, which appear in the Hospital. Other enemies include the Black Maw, eyes of Hell, maggots, Oobs, and zombie meat boys (called burnt boys) from Rapture, as well as several bosses such as Lil' Slugger.

Almost anything that moves or is animated can kill Meat Boy, other than birds, squirrels, bunnies and the other 'cute' animals. The red blobs known as Betus can kill on contact. Saws (of course) can kill on contact, as well as the blood in the hospital and salt in the factory. The purple blobs known as dust bunnies are another thing to avoid. Lava should not be tempered with. Oobs (flying enemies that become more aggressive as you move closer) should be avoided to the best of your ability. Burnt boys (the other meat boys) will explode on touch, and will actively chase the player character once they are sighted. Needles are usually packed in big, gray piles and touching them will kill the player. The laser eyes follow the player, but can be stopped by walls, debris, and other solid objects. Fire balls are found in Chapter 4: Hell, along with portals that shoot black gaping mouths that explode into smaller but still deadly gaping mouths at the player (these enemies are also known as faganzos.) Falling off the screen, be it down, left, right and/or up will also result in death and should be counted as the most deadliest and most helpful enemy (when you get stuck). Other enemies (other than the obvious bosses and Dr. Fetus) include maggots, missile launchers, saw blade lauchers, fire, and fans.