Friday night's vote came after the Transport Workers Union of America, or TWU, leadership struck the deal with Muni management two weeks ago.
It called for using more part-time employees to work during rush hours and it required operators to work 40 hours in a week before they could earn overtime pay.
The plan could have saved $19 million over two years.
The rank and file voted down a similar plan in February.
San Francisco Mayor Gavin Newsom called the rejection "a slap in the face to everyone who rides Muni and to every other public employee union member."