Challenges
| Name | Character | Starting items | Disabled rooms | Curse filters | Additional notes | End goal | Unlock condition | Reward |
|---|---|---|---|---|---|---|---|---|
| 1. Pitch Black | Isaac | - | - | Mom | - |  Hagalaz | ||
| 2. High Brow | Isaac | Butt Bombs
Number One E. Coli Flush! Petrified Poop | - | - | Mom | - |  Jera | |
| 3. Head Trauma | Isaac | Tiny Planet
Soy Milk Iron Bar The Small Rock | - | - | Mom | - |  Ehwaz | |
| 4. Darkness Falls | Eve | Razor Blade
Dark Matter Pentagram Sacrificial Dagger | - | - | Satan | Defeat Mom's Heart 11 times and defeat ??? as Eve. |  Dagaz | |
| 5. The Tank |  Magdalene | Thunder Thighs
Bucket of Lard Infamy | - | - | Mom | Hold 7 Red Heart containers at once. |  Ansuz | |
| 6. Solar System | Isaac | Distant Admiration
Forever alone Halo of Flies Transcendence | - | - | Mom's Heart | Defeat Mom's Heart 3 times. |  Perthro | |
| 7. Suicide King | Lazarus | Ipecac
My Reflection Mr. Mega | - | - | Isaac | Defeat Mom's Heart 11 times and unlock Lazarus. | Suicide King | |
| 8. Cat Got Your Tongue | Isaac | Guppy's Tail
Guppy's Head Guppy's Hairball | - | - | Mom | Obtain the Guppy transformation. |  Algiz | |
| 9. Demo Man | Isaac | Dr. Fetus
Remote Detonator Match Stick | - | - | Mom's Heart | Defeat Mom's Heart 9 times. | Chaos Card | |
| 10. Cursed! | Magdalene | Treasure Map
The Compass Blue Map Raw Liver Child's Heart | - | - | Mom | Hold 7 Red Heart containers at once. | Credit Card | |
| 11. Glass Cannon | Judas | Epic Fetus
Loki's Horns | - | - | Satan | Complete challenge #19: The Family Man, defeat Lokii, and unlock Judas and It Lives! | Rules Card | |
| 12. When Life Gives You Lemons | Isaac | Lemon Mishap
Habit 9 Volt Lemon Party | - | - | Mom | - | A Card Against Humanity | |
| 13. BEANS! | Isaac | Butt Bombs
Pyro The Black Bean The Bean 9 Volt | - | - | Mom | - | Burnt Penny | |
| 14. Its In The Cards | Isaac | Starter Deck
Deck of Cards 9 Volt The Battery Random card | - | - | Mom | - | SMB Super Fan | |
| 15. Slow Roll | Isaac | Polyphemus
Cupid's Arrow My Reflection | - | 0.5 minimum fire rate. damage cannot go below 100. shot speed cannot go over 1.0. | Mom | - | Swallowed Penny | |
| 16. Computer Savy | Isaac | Technology
Technology 2 Spoon Bender | - | - | Mom | - | Robo-Baby 2.0 | |
| 17. WAKA WAKA | Isaac | Strange Attractor
Anti-Gravity | - | - | Mom | - | Death's Touch | |
| 18. The Host | Isaac | The Mulligan
Spiderbaby Tick | - | - | Mom | - | Tech.5 | |
| 19. The Family Man | Isaac | Dad's Key
Sister Maggy Brother Bobby Rotten Baby BFFS! | - | - | Isaac | Obtain both Key Piece 1 and Key Piece 2 in a single run. | Epic Fetus | |
| 20. Purist | Isaac | - | - | - | Mom's Heart | Defeat Mom for the first time. |  Berkano | |
| 21. XXXXXXXXL | Isaac | - | - | - | Every floor is the size of the Womb if it was affected by Curse of the Labyrinth!. However, the floors of each chapter are not combined. | Mom's Heart | Defeat Mom for the first time. |  Gold Heart |
| 22. SPEED! | Isaac | - | - | - | Enemies are sped up as if like under the Broken Watch effect. Isaac moves faster without having an effect on his speed stat. | Mom's Heart | Defeat Mom for the first time. | Get Out Of Jail Free Card |
| 23. Blue Bomber | ??? | Kamikaze!
Mr. Mega Pyromaniac Brother Bobby | - | - | Satan | Destroy 10 Tinted Rocks and defeat Mom's Heart 11 times. |  Golden Bomb | |
| 24. PAY TO PLAY | Isaac | Sack of Pennies
Money = Power | - | +50¢. Every door is an Arcade door that requires 1¢ to open. Taking damage causes Isaac to lose money as if attacked by Greed. Having 0¢ will kill Isaac. | Isaac | Defeat Isaac as Cain. | Percs! + Addicted! | |
| 25. Have a heart | Isaac | Charm of the Vampire
| - | - | All Red Heart pickups (except  Blended Hearts) are replaced with coin pickups respective to the heart's value.
Items that actively or passively heal Red Hearts will no longer do so, including HP up items. | Mom's Heart | Defeat Mom for the first time. | Re-Lax + ??? |
| 26. I RULE! | Isaac | Mom's Knife
Trinity Shield The Boomerang The Ladder Key Piece 1 Key Piece 2 | - | - | Mega Satan | Defeat Mega Satan and unlock The Negative. | Poker Chip | |
| 27. BRAINS! | ??? | Bob's Brain (x3)
Thunder Thighs | - | - | ??? | Defeat Isaac 5 times. | Stud Finder | |
| 28. PRIDE DAY! | Isaac | Rainbow Baby
3 Dollar Bill Rainbow Worm | - | Rainbow Worm cannot be dropped/replaced. | Mom's Heart | Defeat Mom for the first time. | D8 | |
| 29. Onan's Streak | Judas | Chocolate Milk
| - | - | +2 damage. Missing a shot deals half a heart of damage to Judas. | Isaac | Unlock Judas and It Lives! | Kidney Stone |
| 30. The Guardian | Isaac | Punching Bag
Isaac's Heart Holy Grail Spear Of Destiny | - | Isaac's Heart follows Punching Bag rather than Isaac.
Isaac has a unique hairstyle and a reskinned Spear of Destiny. | Mom's Heart | Defeat Mom for the first time. | Blank Rune | |
| 31. Backasswards | Isaac | Key Piece 1
Key Piece 2 8-10 random items | The challenge starts from the Mega Satan fight, and the floors are in reverse order.
Isaac loses a heart container on every floor, as well as the oldest item in his inventory. Every floor starts with a boss fight and ends with a beam of light in the starting room. | Stage 1 | Defeat Mega Satan and unlock The Negative. | Lazarus starts with Anemic. | ||
| 32. Aprils fool | Isaac | - | - | - | Every floor's boss is The Bloat.
Stonies have a chance to be replaced with Cross Stonies. All items, trinkets, and pickups display an incorrect sprite. Active items and pickups always activate a random effect. Isaac's location on the map is displayed as a random room. | Mom's Heart | Defeat Mom for the first time. | Magdalene starts with a Full Health pill. |
| 33. Pokey mans | Isaac | Mom's Eyeshadow
Friendly Ball | - | Friendly Ball is unable to capture monsters, and will instead spawn a random friendly monster. It instantly recharges once the spawned monster dies. | Isaac | Defeat Mom's Heart 11 times. |  Charged Key | |
| 34. Ultra hard | Isaac | Key Piece 1
Key Piece 2 | - | All enemies spawn as champions if possible.
Boss fights will be double trouble if possible. Heart pickups are unable to spawn. | Mega Satan | Defeat Mega Satan and unlock The Negative. |  Samson starts with Child's Heart. | |
| 35. PONG | Isaac | Cupid's Arrow
Rubber Cement | - | 0.2 minimum fire rate. damage cannot go below 100. +10 range. | ??? | Defeat Isaac 5 times. | Greed's Gullet | |
| 36. Scat Man | Isaac | 9 Volt
Skatole The Poop Butt Bombs (x3) BFFS! Thunder Thighs Dirty Mind Mysterious Candy | - | - | Mom | - | Dirty Mind | |
| 37. Bloody Mary |  Bethany | No Book of Virtues
The Book of Belial Blood Bag Anemic Blood Oath Child's Heart | - | - | Satan | Unlock Bethany, Blood Bag, and It Lives! | Sigil of Baphomet | |
| 38. Baptism by Fire | Bethany | No Book of Virtues
Schoolbag Guppy's Paw Urn of Souls Maggy's Faith | - | - | Isaac | Defeat Satan as Bethany, defeat Mom's Heart 11 times, and unlock Maggy's Faith. | Purgatory | |
| 39. Isaac's Awakening | Isaac | Spirit Sword
Mom's Bracelet Trinity Shield | - | - | Mother | Defeat Mother for the first time. | Spirit Sword | |
| 40. Seeing Double | Jacob and Esau | 20/20
20/20 (P2) X-Ray Vision (P2) | - | - | All enemies and bosses are doubled. | Mom's Heart | Defeat Mother for the first time. | Broken Glasses |
| 41. Pica Run | Isaac | Mom's Purse
Marbles Mom's Box | - | - | All items are replaced with trinkets.
Marbles activates on every hit. | Isaac | Defeat Mom's Heart 11 times and unlock Marbles. | Ice Cube |
| 42. Hot Potato | Tainted Forgotten | - | - | The Forgotten explodes every 2.5 seconds. The explosion can deal damage to The Soul. | Satan | Open the hidden closet on the Home floor as The Forgotten. | VII - The Chariot? | |
| 43. Cantripped! | Tainted Cain | - | - | All cards and pills are replaced with Item Cards.
Bag of Crafting initially has 3 card slots. Every item is replaced by Starter Deck which grants +1 card slot and spawns 3 Item Cards. | Mom | Open the hidden closet on the Home floor as Cain. | VIII - Justice? | |
| 44. Red Redemption | Tainted Jacob | Dad's Key
| - | - | The starting room has no rooms connected to it. Rooms spawn in clusters around the map must be found via Red Key.
Red Key has infinite uses but after 20 uses Dark Esau will spawn. Each subsequent 5 uses will spawn another Dark Esau if Tainted Jacob has not turned into his Lost form. I AM ERROR rooms cannot be accessed via Red Key. Every floor generates as a Curse of the Labyrinth! floor, having 1 Shop, 2 Treasure Rooms, and 2 Boss Rooms. The 4th floor has only 1 Boss Room. Any room may be from any floor of the current chapter. | Mother | Open the hidden closet on the Home floor as Jacob and Esau. | IX - The Hermit? |
| 45. DELETE THIS | Isaac | TMTRAINER
| - | - | Each area's theme, obstacles, floor texture, and music, are randomized, and can sometimes glitch out.
Shop prices vary from 1¢ to 99¢. The challenge has no name and appears invisible in the Challenge List. | ??? | Unlock Tainted Eden. | XIV - Temperance? |