There are three types of flies in the game (four with the Wrath of the Lamb DLC).
Black flies buzz around the room randomly, often moving away from the player. They cannot damage Isaac at all, even by touch, making them completely harmless. However, they can turn into relatively dangerous Pooters if they fly around Poop for too long.
Attack Flies are red and black flashing flies that will follow Isaac relentlessly until killed. Their only way of damaging Isaac is by contact, and they move rather slowly, making them easy to kill. However, they can be dangerous when found in groups, as they can swarm around Isaac quickly.
Attack Flies turn into regular Black Flies if Isaac has Skatole. They take a small amount of time to convert into Black Flies, so it is possible for Isaac to be damaged if the player is not careful.
Large Attack Fly
These have the exact same behavior as regular attack flies, and move at the same speed. However, they're larger and have more health than regular Attack Flies.
Large Attack Flies are unaffected by Skatole.
Moters appear as two Attack Flies joined together. They behave similarly to regular Attack Flies, but split into two Attack Flies upon being killed. Their split is very wide and unpredictable and can easily shave off half a heart. It is recommended to kill faster flies and other small monsters before it as the two flies it spawns are actually more dangerous in many ways. Running between them just as they split is also a very good way to dodge.
If Isaac has Skatole, killing a Moter will not make them split into two Attack Flies.
Blue flies spawn from triggering the effect of Infestation, The Mulligan, and Guppy's Head. They orbit around Isaac and follow him throughout rooms. Blue flies charge at nearby enemies and die upon impact. The blue fly from the item Forever Alone is an Orbital Familiar that acts entirely differently and does not charge.
The Eternal Fly is a white Fly that was added in the Wrath of the Lamb DLC. They can be found on any floor in the game. They usually circle Corn Poop or enemies like Holy Leeches and the Hanger and will not stop circling them until their hosts are destroyed. Before that, they will deal damage to Isaac if he touches them and are completely immune to all forms of damage. When their hosts are destroyed, they convert into Large Attack Flies with increased health.
If Eternal Flies are circling Corn Poop and there are no other enemies around, you can leave the room at will. This is true even if they have already been converted into large attack flies: they will simply disappear when you return into the room.
- If the host of an Eternal Fly is destroyed while it is frozen due to the use of items such as Mom's Bra, the fly will simply sit in the same spot without doing anything, being pushed by any tears shot at it.