Jose Manuel Maza said that any of the municipal leaders who agreed to help stage next month's vote should be arrested if they fail to appear.
Prosecutors earlier ordered the seizure of ballot papers and voting materials.
Catalonia's vote on breaking away from Spain is deemed illegal and has been suspended by the constitutional court.
But Catalonia's pro-independence government has said that the referendum, which is planned for 1 October, will go ahead.
If the vote is held, prosecutors could charge the council leaders with misuse of public funds.
The Association of Municipalities for Independence has written to hundreds of Catalan officials assuring them that they are doing nothing wrong in helping to run the ballot, despite such actions defying Spain's constitutional court.
In response to the Spanish government's latest move, the Catalan authorities have urged the region's mayors to take to the streets of Barcelona in protest on Saturday.
On Tuesday, the Spanish Public Prosecutor's Office instructed security forces to remove all voting materials which it said could help with the "consummation of the crime".
The order included promotional materials and the ballots themselves, Spanish newspaper El Pais reported [in Spanish].
With their own language and customs, Catalans already have much autonomy, but opinion polls suggest that the vote, if it takes place, will be very close.
There is a widespread feeling in the region - one of Spain's richest - that too much of its tax revenue goes to Madrid.
Meanwhile Spanish Prime Minister Mariano Rajoy and his conservative Popular Party have vowed to do everything within their power to stop the referendum, which they argue cannot take place according to Spain's 1978 constitution.
On Wednesday, the official referendum website was shut down following a court order, Spain's Guardia Civil police said.