There are three new playable characters in Antibirth, Bethany, Jacob & Esau and Edith, each with a unique playstyle.
| Character |  Bethany |
  Jacob & Esau |
|---|---|---|
| Description | Any Soul Hearts, Black Hearts, and Immortal Hearts that Bethany picks up are added to a counter rather than to her health bar. Each half heart in the counter can be consumed to contribute a point of charge to her currently held active item. | Both Jacob and Esau are controlled simultaneously. Their health and items are independent, but if one of the two dies, both die. |
 Health
|
|
Jacob: Esau: 
|
 Damage
|
3.5 (*1.00) | Jacob: 2.75 (*1.00) Esau: 3.75 (*1.00) |
 Tears
|
10 | Jacob: 9 Esau: 11 |
 Shot Speed
|
1.00 | Jacob: 0.85 Esau: 1.15 |
 Range
|
15.75 | Jacob: 23.75 Esau: 23.75 |
 Speed
|
0.85 | Jacob: 1.00 Esau: 1.00 |
 Luck
|
0 | Jacob: 1 Esau: -1 |
 Starting Pickups
|
None | None |
| Starting Item(s)
|
Book Of Virtues | None |
| Unlockables | ||
| Boss Rush | Beth's Faith | Rock Bottom |
| Mom's Heart (on Hard) | Soul Baby | Double Baby |
| Sheol | Urn Of Souls | Red Stew |
| Cathedral | Book Of Virtues | Jacob's Ladder |
| The Dark Room | Alabaster Box | Damocles |
| The Chest | Blessed Penny | Birthright |
| The Witness | Nothing (currently bugged) | Magic Skin |
Unlocking
Each character is unlocked by accomplishing a certain task in the game. The unlock requirements are as follows:
| Character | Unlock requirement |
|---|---|
| Bethany | Have 12 Soul Hearts at once. |
| Jacob & Esau | Defeat The Witness. |
Notes
- A third new character, Edith, was cut due to balancing/design issues.