The move increases the pressure on Catalan officials just one day before the pro-separatist camp officially kicks off its referendum campaign in the Mediterranean port of Tarragona.
Furious at the decision to instigate a probe, Catalan municipal associations called on all the region's mayors to protest in Barcelona on Saturday to show their "rejection of a Spanish judicial system that goes after the media, ballot papers, ballot boxes... and now mayors".
The prosecutors' office ordered the mayors who have agreed to help stage the vote be summoned to court as official suspects and if they do not appear to "order their arrest", according to a copy of the ruling obtained by AFP.
Pro-independence Catalan president Carles Puigdemont dubbed the move an "atrocity scarcely worthy of a democracy", and said he would attend Saturday's protest.
Catalonia's pro-separatist government has asked the wealthy northeastern region's 948 mayors to provide facilities for polling stations for the plebiscite.
So far 712 mostly smaller municipalities have agreed to participate, according to a list posted on the website of Catalonia's Municipal Association for Independence (AMI) which represents 750 municipalities.