The cannabis clampdown comes just days after the first closure of a club for suspected illegal dealing.
Cannabis clubs in the city have fuelled a boom in tourists looking for legal highs, despite technically being private establishments meant for members only.
It is illegal in Spain to traffic cannabis or smoke it in public but private growing and use are allowed.
Clubs which are run as not-for-profit cooperatives can use this loophole to grow their own marijuana and provide a venue to smoke it.
Some, however, have been accused of bending the rules by buying cannabis on the black market and enticing tourists to join.
This has prompted the city council, which has traditionally tolerated the clubs as long as they kept to the rules, to crack down on the reefer-related wrongdoing.
Barcelona's deputy mayor, Joaquim Forn, said, "We cannot allow there to be those who, in the guise of businesses, threaten the coexistence, health and wellbeing of the people."
He underlined that associations for the supply and consumption of cannabis could not be based in public places, nor use publicity, nor encourage consumption, according to Spanish daily 20 Minutos.
He also added that the local government was worried about Barcelona's burgeoning reputation as a destination for cannabis tourism and noted that they were more concerned with health, especially "the effect of drugs on young people".
The future of cannabis smoking associations may hang in the balance as the government reviews their operations but for now at least the city's 160 clubs are safe.
"The law allows, in very specific cases, the consumption of marijuana and this is for residents as well as tourists," Forn said.