The environmental group Sea Shepherd said fishermen fired 25 shots at one of its night-vision drones in Mexico's Gulf of California, bringing it down.
Various drones have been employed to patrol the Gulf, also known as the Sea of Cortez, to combat illegal fishing and save the critically endangered vaquita marina, the world's smallest porpoise.
Poachers often go out at night to set nets for totoaba, a fish whose swim bladder is prized in China. But vaquitas often get caught in totoaba nets, causing the population to plunge to less than 30.
Sea Shepherd has been the target of demonstrations by fishermen in the past, but said the Christmas Eve shooting represented "a new level of violence."
The group said Tuesday that its drone had located four small boats illegally fishing for totoaba.
Men on three of the boats were observed firing at the device until its camera shut off.
The drone was then listed as "disconnected," indicating it went down.
In the past, fishermen have thrown rocks and bricks at drones, staged demonstrations demanding that Sea Shepherd boats be expelled, burned vehicles and patrol boats, and beat inspectors from the office of environmental protection, but this is the first time they fired guns.
In other parts of the world, Sea Shepherd vessels have rammed into whaling ships to deter illegal activities.
But in the Gulf, the group has peacefully patrolled the waters, looking for vaquitas dead or alive and gill nets, which it removes.
The patrol effort has been welcomed by the Mexican government, which has had a difficult time enforcing a ban on gill net fishing because fishermen use fast boats, leading vessels on hours-long chases.
Sometimes, pickup trucks drop boat trailers onto beaches and haul off small fishing crafts before authorities arrive.