The bees entered the hive through a hole in a carved flower crafted by the chapel’s master stone masons.
The 15th Century Midlothian building is undergoing a £13m conservation and site improvement project.
The discovery was made when two pinnacles, which had been made unstable by nesting jackdaws, had to be taken down stone by stone and rebuilt.
Malcolm Mitchell, of Page Park Architects, said: “It was a big hollow about the size of a gas cylinder and the hive had obviously been abandoned.”
It is believed that the bees left the hive when a canopy was put over the chapel during renovation works. Another pinnacle had a similar hollow, but no access hole.
“Master masons built these in, whether it was under direction or not. What you find at Rosslyn is there are so many irregularities and nuances in the stone work and it’s as if the stone masons are teasing us from the past,” Mr Mitchell said.
“These hives were never intended to be a source of honey. They were there purely to protect the bees from our inclement weather.”