The new rule restricts religious or political speech after dark on the popular street.
Assembly of God Pastor Paul Gros told CBN.com that police asked him to leave or face arrest while he preached on Bourbon Street with his wife back in May.
Pastor Troy Bohn of Raven Ministries, told WWLTV: "We've really had no issues whatsoever, we've seen God do some tremendous things and have not even had issues with law enforcement."
"Our team is where its been for the last two years, three nights a week, talking to people, sharing the gospel under the red cross. The police approached us and told us we were under arrest."
During this year's Southern Decadence festival, religious groups were arrested and charged with aggressive solicitation by the police.
The pastors are fighting back by taking the city to court with help of the ACLU.
ACLU of Louisiana Senior Staff Attorney Justin Harrison told WWLTV: "All speech on Bourbon Street between the hours of sunset and sunrise of a social, political or religious nature. Its the religious nature that brought the ordinance to our attention in this case."
Harrison says a judge approved a temporary restraining order against Mayor Mitch Landrieu, the City of New Orleans and NOPD Superintendent Ronal Serpas. The next step is a pending lawsuit citing freedom of speech rights.
However, not everyone is happy. Mary Griggs, Director of the Lesbian and Gay Community Center of New Orleans, says religious groups with bullhorns go against the spirit of Bourbon Street: "I certainly have issues with their argument. I don't think that freedom of speech should trump my right to life, liberty and the pursuit of happiness."
"I think that's not appropriate for the space that they're in. If they want to witness about Jesus Christ there are certainly other appropriate places that they can be."