Craig and Bradley Barnett left the six live hens in the hotel room in Bournemouth, Dorset, as a joke but later that evening the party set the hens free.
As a result of being released, one of the chickens was slaughtered by a fox and a second was later found dead.
Two others were found in a terrified state the next morning by RSPCA inspector Graham Hammond. The other two were never seen again.
Hotel's CCTV shows the Barnett brothers taking the hens up to the stag's room. The video later showed at least two friends dressed in their boxer shorts carrying the birds back down and releasing them in the car park at 3.40am.