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.
Craig Barnett, 23, and Bradley Barnett, 21, were prosecuted by the RSPCA under the Animal Welfare Act 2006.
Magistrates heard that the pair were part of a dozen-strong stag party that visited Bournemouth, Dorset, on July 22 last year.
On their way to the party town, the Barnetts stopped off at a farm in the New Forest and bought the brood of hens.
They drove to a hotel in Bournemouth where they left the birds in cardboard boxes in the back of their car for five hours while the group went to a greyhound stadium.
When they returned at 9.12pm the Barnetts carried the birds in the boxes into the hotel room and left them in the bathroom.
Matthew Knight, prosecuting for the RSPCA, said: "In their interviews, the Barnett brothers stated they left the chickens in the hotel room as a surprise for the stag.
"They said it was a case of members of the stag party egging each other on.
"Some members of the stag party returned to the room at 3.30am and found the chickens and the mess they had left in the bathroom.
"CCTV footage showed a number of young gentlemen in their boxer shorts carrying the chickens downstairs to the lobby area and outside.
"The RSPCA inspector found one chicken had been taken by a fox and one was found dead. Two more were found and later rehomed and two were never found."
Mr Knight said as well as being left in the hot and cramped boxes, no food had been left for the birds in the room and, although the bathroom sink was filled with water, the hens could not reach it.
He added: "To leave the chickens without food or water for 14 hours was unacceptable.
"Using animals for the purposes of entertainment or a prank has long been a criminal offence and socially unacceptable in this country."
Nigel Ley, defending both Barnett brothers, said the pair had bought the chickens as a present for their mother who runs a farm in Horsham, West Sussex.
He said: "At the time they thought it was an acceptable thing to do. They now realise what a stupid thing this was. It was done in a moment of absolute stupidity.
"They assumed if the chickens were left in a hotel room with the freedom to fly around they would be quite happy."
Magistrates in Bournemouth fined Bradley Barnett, a university student from Horsham, £110 pounds with a £15 victim surcharge.
Craig Barnett, a salesman for a communication company from Sherborne, Dorset, was fined £300 with a £15 victim surcharge.
They were also ordered to pay £375 costs each.
Francis Vine, chairman of the bench, said: "This started out as a foolish prank which went wrong and you are fully aware of the consequences of this type of behaviour."
The bench did not impose an animal banning order on the pair as they accepted they were unlikely to repeat the prank again.
The Barnett brothers pleaded guilty at an earlier hearing to a charge of failing to provide a suitable environment for the chickens.
