The order was issued by Annise Parker, the city's first openly lesbian mayor. Any pastor who refuses to obey could be held in contempt of court.
Alliance Defending Freedom attorneys have now filled a complaint about the plans to a Texas court.
The organisation claims the new rules have been introduced so that the authorities can find out if the pastors have ever opposed or criticised the city.
ADF senior legal counsel Erik Stanley said: "City council members are supposed to be public servants, not 'Big Brother' overlords who will tolerate no dissent or challenge.
"In this case, they have embarked upon a witch hunt, and we are asking the court to put a stop to it."
The new rules are part of Houston's non-discrimination ordinance, which, among other things, would allow men to use ladies' toilets and vice versa.
This legislation was objected to by the group of pastors, who were in a coalition of 400 Houston-area churches that opposed the ordinance.
"The city's subpoena of sermons and other pastoral communications is both needless and unprecedented," said ADF litigation counsel Christiana Holcomb.
"The city council and its attorneys are engaging in an inquisition designed to stifle any critique of its actions. Political and social commentary is not a crime; it is protected by the First Amendment," she added.
Fox News reports that City Hall refused to comment on the situation.