Airspace over the capital was closed to drones at the start of the Catholic Holy Year, or Jubilee, earlier this month over fears the remote-controlled aircraft could be used in a terrorist attack.
Police patrolling the Vatican area spotted the drone, which contained a high resolution camera, flying over the Tiber River and St Peter's Square on Sunday morning, Roma Today reported.
The police found the men, identified as being Israeli, on a bridge near St Peter's. One of them was holding the drone's remote control.
The men are being questioned and will likely face charges for violating the ban.
Security forces have been put on high alert due to fears of jihadist attacks like the Paris carnage of November 13th.
The no-fly zone, which also applies to regular aircraft, is expected to stay in place for the rest of the Holy Year, which ends in November next year.