Treasure Rooms will always contain one item from the Treasure Room pool, and never contain enemies in Rebirth. However, they may contain pots or skulls, which have a chance to spawn enemies when broken.
Treasure Rooms never border more than a single regular room, but there may be a Secret Room adjacent to it.
There's a rare chance that a treasure room might contains two items to choose from. Golden Horse Shoe will increase the chance for a treasure room to become a double treasure room, and More Options will cause every treasure room to become a double treasure room.
There is a chance that a treasure room item will be surrounded by spikes or rocks, requiring Isaac to use a bomb or sacrifice health to reach the item.
The Treasure Room may contain 3 black flies.
The Treasure Room may contain a Restock Box, allowing Isaac to reroll offered items if he has coins or bombs.