The Duke of Flies constantly floats around the room in diagonal directions. Knockback from tears and other sources can cause him to change travel direction. He has three attacks that he can perform while he floats:
- Spawns 3 Attack Flies that will orbit around him, blocking shots.
- Spawns a Large Attack Fly that will chase Isaac.
- Sends any orbital flies outward to chase Isaac.
- He can only perform this attack if he has 5-6 orbiting flies.
- If he has 4 orbiting flies he cannot perform this attack nor can he spawn more orbiting flies as it would exceed his limit resulting in him only spawning chasing Attack Flies.
- There is a total limit of 12 flies of either type alive at once and The Duke of Flies can never spawn more to exceed this limit.
- Upon death, all orbiting flies will be free to chase Isaac.
- Spawns 6 random Flies or Attack Flies upon death.
- Unlike regular Attack Flies the ones spawned by The Duke of Flies can be killed if Isaac walks into them though they will still deal contact damage before dying.
- This does not include the Attack Flies spawn upon his death.
Champion Versions 
- Green: Spawns Moters instead of Large Attack Flies and shoots blood shots in eight directions when spreading the orbiting flies. He can spread the Attack Flies at any moment instead of only performing it when having enough of them orbiting him, similar to The Husk.
- Orange: 15% larger than normal with 25% more HP (137.5) and spawns Suckers instead of Large Attack Flies.
The Duke of Flies as one of Delirium's forms.
- If Isaac has the Beelzebub transformation, the Attack Flies that the Duke of Flies spawn will instantly turn into blue flies which damage him.
- The Duke of Flies appeared in the original The Binding of Isaac.
- In the original Binding of Isaac, the flies in The Duke of Flies's mouth were animated, this was removed in Rebirth but readded in Repentance.
- The Duke of Flies seems to be a human corpse in the bloating stage of decomposition, filled to the brim with flies. The Duke has bloated to the point that it has lost its once human silhouette, becoming nothing more than a sphere with dangling nubs that used to be its appendages.
- Any enemy summoned by The Duke of Flies dies immediately upon making contact with Isaac.
He is the boss version of the Dukies.
- The Husk is the posthumous version of him.
- The stitching under The Duke of Flies' mouth is an autopsy stitch, an incision in the shape of a Y. The incision allows passage to the cadaver's insides; when the autopsy is complete, it is stitched back up.
- The name "The Duke of Flies" is a reference to the demon Beelzebub, and an alternate name for the devil who is known by the title "Lord of the Flies".
- The name could also be a reference to the book "Lord of the Flies", specifically the character named Lord of the Flies - a severed pig's head mounted on a pike, swarming with flies.
- When under the effects of Curse of Darkness, a brief flash of light is seen when The Duke of Flies summons flies.
- Edmund McMillen has confirmed The Duke Of Flies to be his favorite boss design in The Binding Of Isaac in an AMA thread.
- In another AMA for the Binding of Isaac subreddit, Edmund had also confirmed The Duke Of Flies to be a super old design, before Meat Boy in 2007. However, he never used the design until developing The Binding Of Isaac.
- The Duke of Flies additionally returns in the Binding of Isaac spinoff, The Legend of Bum-bo, under the simpler name of "The Duke", which further concretes him as being special to Edmund.
- In Repentance, The Duke Of Flies resembles his sprite from the original Binding of Isaac.
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