The governor's comments came during a press conference in Fort Myers on monoclonal antibody treatments, which the governor has worked to make more accessible in the state to prevent severe COVID-19 infections.
"I think that the mandate is going to lose in court," DeSantis said during the press conference regarding the federal mandate.
He added that Florida would contest any such mandate from the Biden administration "immediately," and that any lawsuits from the state would be filed in the 11th U.S. Circuit Court of Appeals.
"We think the state of Florida has standing to do it," he said. "We also know businesses that we’re going to work with to contest it."
The governor also said Florida has "a responsibility at the state level to do whatever we need to do to protect Floridians from mandates that could result in them losing their jobs."
DeSantis and Biden have taken opposite stances on vaccine and mask mandates. While the president issued a mask mandate on federal grounds soon after taking office, DeSantis barred local mask mandates in Florida.
Last month, the president announced businesses with more than 100 employees will be required to mandate COVID-19 vaccines or administer weekly tests. Employers are also required to pay employees for time off to get vaccinated and recover from side effects.
The rule is expected to take effect once the Office of Management and Budget (OMB) completes its review of the Occupational Safety and Health Administration's (OSHA) initial text of an emergency vaccine rule.
OSHA "has been working expeditiously to develop an emergency temporary standard that covers employers with 100 or more employees to ensure their workers are fully vaccinated or undergo weekly testing to protect employees from the spread of coronavirus in the workplace," a Department of Labor spokesperson said in a Wednesday statement.
The Department added that on Tuesday, "as part of the regulatory review process," OSHA submitted "the initial text of the emergency temporary standard to the Office of Management and Budget."
Nearly 70% of U.S. adults have received both vaccine doses, while more than 60% off all Americans have received at least the first dose.
In June, 99.5% of all COVID-19 deaths were of unvaccinated people. There have been a total of 14,115 U.S. deaths as a result of breakthrough cases, or COVID-19 cases that occurred in vaccinated individuals, and 70% of those vaccinated individuals were older than 65. The Centers for Disease Control and Prevention (CDC) has noted that no COVID-19 vaccine is 100% effective against the virus.
Fox News' Brie Stimson contributed to this report.