If a non-marked enemy is killed, the skull will disappear and no bonuses will be granted for that room.
Notes
Losing this item will not remove stat rewards.
As long as the room is not cleared, Isaac can re-attempt a Death's List by leaving and re-entering.
The marked enemies are chosen in the same order in a given room, starting from the top-left corner with enemies that are vulnerable at their spawn time (from top to bottom, then from left to right). If new enemies are spawned via Portals or on death, they are added to the end of the marked queue.
The marked Army Fly will always be the last one in the line of Army Flies.
Bishops can shield the marked enemy unless frozen or petrified beforehand.
Killing all piles of a Cursed Globins counts as one kill.
A room with no enemies that instead requires Pressure Plates to be pressed will also give a reward when cleared.
If a reward stat is chosen that is already at maximum, the sound effect and Isaac's thumbs up animation will play, but there is no other effect.
Bosses may also be marked.
If a Portal is marked and despawns, no other enemy will be marked and the bonus is lost.
Enemies that are not spawned when the room is entered (such as Angels from bombed Angel Statues and enemies in Challenge Rooms, Greed Mode, and Boss Rush) will not be marked.
In the Mom boss fight, each of Mom's body parts (eyes, foot, and hands) is considered as a different enemy, and Isaac has to kill the foot to get the bonus.
If the doors open automatically due to spending a long enough time in the room, any existing mark will disappear.
Familiars, especially ones that passively or actively damage enemies of their own accord, may make killing enemies in order difficult, if not impossible.
Ace cards will kill the enemies in the correct order when possible.
If a Mr. Maw is marked, killing either part of its body will not forfeit Death's List, as the remaining enemy of said Mr. Maw will still be marked.
This item has little to no effect in Greed Mode.
Waiting for the Room Anti-softlock to trigger will always grant a reward from Death's List.
Friendly enemies cannot be marked, and their deaths do not cause the mark to disappear.
Synergies
Ace Cards: Enemies that die by being turned into pickups don't disrupt the Death's List chain and clearing a room by turning all its enemies into pickups still grants Isaac the Death's List reward.
Door Stop: If Isaac has failed Death's List but hasn't cleared the room, leaving and re-entering will reset the list.
E. Coli/Flush!: Enemies that die by being turned into poop do not disrupt the Death's List chain and clearing a room by turning all its enemies into poop still grants Isaac the Death's List reward.
Eraser: Erasing enemies makes it easier to complete the list as erased enemies do not disrupt the Death’s List chain and rooms cleared by Eraser’s effect will also reward the bonus.
Interactions
Freezing attacks: Enemies that die by freezing do not disrupt the Death's List chain and clearing a room by freezing its enemies still grants Isaac the Death's List reward, though the shards caused by them breaking do.
Toxic Shock: Not recommended. If multiple enemies are killed by Toxic Shock at the same time, the mark will disappear and no reward will be given, even if the enemy next in line to be marked was killed at the same time as the marked one.
Swarmers will lose the mark and no reward will be granted.
Bug!
Multisegmented enemies such as Mr. Maw and Pin may be impossible to kill in the correct order, as each segment is considered a separate entity and are destroyed as the enemy on the whole dies.
Bug!
Other multisegmented enemies such as Larry Jr. can have their segments destroyed in a proper order, but the skull marker is displayed on the wrong segment.