Bosses are very strong monsters found in boss rooms at the end of a floor. The boss at the end of the floor must be defeated in order to advance to the next floor. In later chapters, bosses are able to spawn as degraded bosses, or normal enemies. Same can go for mini-bosses, which can spawn on later floors as degraded mini-bosses. This starts in the caves, where the Gurglings are often encountered. The boss for a particular floor is chosen from the pool of bosses for the Chapters of the floor. Bosses may also spawn as champions, which can change various attributes such as health, damage, and attack pattern. Upon defeat, most bosses will drop an item from the boss item pool.
Bosses boast additional resistances to Status Effects. For example, fear occurs less often and the Night LightA cone of light shines in the direction Isaac is moving that slows enemies and enemy shots. is unable to slow them down.
Additionally, some floors may spawn a room with a Mini-boss.
In total, there are 72 bosses and 18 mini-bosses. 10 bosses were added in Afterbirth and 6 were added in Afterbirth †.
- Originally every boss and enemy in Isaac was going to resemble Isaac in some way.  This is still the case for some.
- Double Trouble boss pools can have bosses from previous chapters, and can be the same for both versions of that chapter's floor. This means that, for example, Double Trouble boss fights in the Caves can feature bosses from the Cellar or the Basement, and every Double Trouble fight in the Caves can also be encountered in the Catacombs.
- Bosses often come in three versions: normal (For example, Monstro), posthumous (Monstro II), and alternate (Gish). Usually, the posthumous boss looks like the original with some horrible mutilation; for example, Monstro and Chub had most of their skin ripped off.
- Most bosses and their alternate/posthumous forms either share similar death animations or share the fact that they lack death animations.
- Some bosses appear as evolved or larger versions of enemies. (For example, Chub looks like a bigger Charger) Some even act as larger versions of other bosses. (Little Horn and Big Horn )