The move ratchets up tension in a struggle over the vote, the day after the Constitutional Court suspended the second of two laws paving the way for the controversial ballot.
And if the mayors do not answer the summons police should arrest them, the prosecutor said in a letter delivered to local authorities on Wednesday.
So far, 712 municipal leaders have pledged to allow the use of public space for the October 1 vote, although the mayor of the area’s main city Barcelona has not taken a definitive position.
Officials engaging in any preparations for the vote could face charges of civil disobedience, abuse of office and misuse of public funds, the letter said.
Catalonia’s regional parliament passed laws last week to prepare for an independence referendum on October 1.
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Spain’s Constitutional Court suspended the vote after Prime Minister Mariano Rajoy challenged it in the courts.
Prime Minister Mariano Rajoy’s government is increasing the pressure to prevent an independence referendum from going ahead.
It has declared the vote illegal and challenged laws linked to the ballot in the courts.
Judges are now considering whether the legislation contravenes Spain’s constitution, which states the country is indivisible.
The Catalan parliament approved both laws on Wednesday in a move which brought a long-running tussle between the pro-independence regional government and the Madrid-based central government to a head.
Barcelona’s Mayor Ada Colau has asked for reassurances that civil servants involved in the process will not risk losing their jobs.
Hundreds of thousands of people took to the streets of Barcelona this week to show support for independence.
Polls have shown support for independence waning in recent years with those wanting a separate state in a minority. However, a majority of Catalans want to vote on the issue.
In a separate order, the Constitutional Court told regional government officials on Wednesday they had 48 hours to show how they were preventing the vote from going ahead.
Catalan leader Carles Puigdemont, who faces criminal charges for his role in organising the referendum, only has the power to call an election not a referendum, a government spokesman said on Tuesday.
A government spokesman and Minister of Education, Culture and Sport Inigo Mendez de Vigo told Antena 3 television: “If Puigdemont wants to consult the people, it’s very simple, he should call an election.”