(Not to be confused with the Challenge Room)
To enter you must have one red heart or less remaining.
Boss Challenge Rooms take on the appearance of a regular Challenge Room on the inside and can be found in second to ninth floors (However it can be found on the first floor, it is unknown if this is a bug). It offers an item which is normally a drop from common bosses. Upon picking up an item, you have to fight two waves of bosses. In the earlier floors, you have to fight Monstro followed by 3 short Larry Jr.s. In intermediate floors, you have to fight Chub and then Peep. In the later floors, the bosses are Loki and Monstro II.
The door to the room has a skull overlapping the two swords. On the map, the logo is a bloody sword.
- ??? always has access to the boss challenge room.
- As with regular Challenge Rooms, if the room is adjacent to a secret room, you may also enter it by destroying the wall that separates the rooms. Notice however that, unless you have XRay Vision, the wall will close again when the fight begins.
- If an activated item is used a split-second before the first boss spawns at the beginning of a Boss Challenge Room, there is a chance that both the first and second boss will spawn simultaneously and you will have to fight them both.
- If you pause the game right after picking up the upgrades and if you wait so that you hear 2-3 poofing sounds which monsters make when they spawn, all of the bosses will spawn at the same time.
- The first wave will also spawn if you pick up an item spawned by a Blood Donation Machine or a Beggar.