Upon activation, all closed doors in the current room Isaac is in will be opened; this includes Challenge Rooms, rooms in The Chest, and even rooms where there are bosses and enemies (except for the fight with Mom.) The item does not, however, open rooms which require a normal key to open, such as Shops, Treasure Rooms, or Libraries.
When activated in the moment an item drops in a room, it will cause an additional item drop. 
- This may indicate that Isaac's father was kind or at least protected Isaac from things, being able to help him escape in-game.
- This could also symbolize how Isaac's father left him and his mother, in much the same way it allows Isaac to leave rooms.
- If used in a Boss Room before the boss has been defeated, Dad's Key will open the Devil Room/Angel Room.
- This does not work on floors where the Devil/Angel Room cannot open by defeating the boss, such as Basement/Cellar 1, Cathedral, The Chest, Sheol, or Depths/Necropolis 2.
- When used in a Challenge Room, opening the door and leaving while the fight is on will continue the music even after you leave until the clip ends.