The workers were removed in favor of cheaper and faster electronic transponders, and a camera system that photographs every license plate that comes through, mailing an invoice to each motorist who doesn't prepay.
Those who fail to pay will receive warnings and could eventually have a hold placed on their vehicle registration at the California Department of Motor Vehicles.
In addition to saving money, the move is expected to improve traffic flow on the iconic span that opened in 1937. The California Highway Patrol said traffic was flowing smoothly, with no delays early Wednesday.
On Tuesday, when their final shifts working on the bridge ended, the toll collectors said their goodbyes. They forced their mouths into smiles, hugged each other tightly and cried as they left their booths for the last time.
Some were angry and said their contribution - helping people with directions, giving a warm greeting to a regular commuter - will be missed.
"Our DNA is embedded in this bridge ... we are part of it," said Jacquie Dean, a career toll collector who had worked on the burnt-orange span for 18 years before her last shift Tuesday. "Some customers still want to pay cash. They don't want to be tracked and photographed."
Many drivers have switched to the FasTrak devices that attach to a car's windshield.
The switchover is expected to save about $16 million in salaries and benefits over eight years. Nine toll takers will lose their jobs. Another 17 have either been placed in other district positions or have retired, Currie said.
"It was a difficult decision and involved the loss of some very dedicated staff," said Mary Currie, spokeswoman for the Golden Gate Bridge Highway and Transportation District.
Employees who loved working on the bridge said the job was something they'd planned on keeping until retirement.
"I never thought that I would ever end my career at the bridge," Dawnette Reed, 43, who started working in the bridge gift shop at 16 and, after a stint in the U.S. Army, became a toll collector at age 26.
"The bridge won't be the same without us."