The 52-48 vote sends the legislation to the Democratic-led House of Representatives, where it faces strong headwinds, while Biden has threatened to veto it.
The legislation would overturn administration rules ordering businesses with 100 workers or more to require vaccinations or coronavirus testing for millions of employees.
The initiative underscores the intense political opposition facing administration efforts to combat COVID-19 at a time when health officials are trying to contain the highly contagious Omicron variant.
"The federal government has no authority to make anyone choose between getting a vaccine and keeping their job," argued Senator Mike Braun, a Republican from Indiana.
Meanwhile Democratic Senator Patty Murray from Washington asked "How on earth does it make sense right now to undercut one of the strongest tools that we have to get people vaccinated and stop this virus? In what world is that a good idea?"