Dave Blondin reopened Prime Fitness & Nutrition in Oxford last month during Phase 1 of the state's plan to resume daily life. Gyms are permitted to operate under Phase 3, which won't begin until the end of June.
He said the gym has been operating at 50 percent capacity.
An attorney for the city told WCVB-TV that the locks to the gym were changed in cooperation with the building's owner.
"The town is hopeful that this action will resolve the matter and assure protection of public health and safety, which is the sole intent of the town in this action," the attorney told the news outlet.
The town shut off the gym's access to water and electricity on Wednesday but members still showed up for their workouts in the dark, the Boston Herald reported.
“If I have to sit in jail to protect my business, then I will," Blondin told Boston 25 News last week. "I’ve literally built this gym since I was 20 years old, I put every little ounce of me into this place and I will not let it go down.”
Messages to the gym from Fox News were not immediately returned.
Blondin's defiance has triggered a battle with town officials, who have fined him more than $8,000 and taken him to court. A Worcester Superior Court judge ruled this week that the town could shut off the gym's utilities.
Blondin said he's planning a protest Saturday in the gym's parking lot and will set up exercise equipment outside so people can workout.