US District Judge Terry Doughty in Monroe, Louisiana, temporarily blocked the Centres for Medicare & Medicaid Services (CMS) from enforcing its vaccine mandate for healthcare workers until the court can resolve legal challenges.
Doughty's ruling applied nationwide, except in 10 states where the CMS was already prevented from enforcing the rule due to a prior order from a federal judge in St Louis.
Doughty said the CMS lacked the authority to issue a vaccine mandate that would require more than 2 million unvaccinated healthcare workers to get a coronavirus shot.
"There is no question that mandating a vaccine to 10.3 million healthcare workers is something that should be done by Congress, not a government agency," wrote Doughty.
Separately, US District Judge Gregory Van Tatenhove in Frankfort, Kentucky, blocked the administration from enforcing a regulation that new government contracts must include clauses requiring that contractors' employees get vaccinated.
The contractor ruling applied in the three states that had filed the lawsuit, Kentucky, Ohio and Tennessee, one of at least 13 legal challenges nationwide against the regulation. It appears to be the first ruling against the contractor vaccine mandate.
The White House declined to comment.
The legal setbacks for President Joe Biden's vaccine policy come as concerns that the Omicron coronavirus variant could trigger a new wave of infections and curtail travel and economic activity across the globe.
Biden unveiled regulations in September to increase the US adult vaccination rate beyond the current 71% as a way of fighting the pandemic, which has killed more than 750,000 Americans and weighed on the economy.
Republican state attorneys general, conservative groups and trade organisations have sued to stop the regulations.
Tuesday's rulings add to a string of court losses for the Biden administration over its COVID-19 policies.
The most sweeping regulation, a workplace vaccine-or-testing mandate for businesses with at least 100 employees, was temporarily blocked by a federal appeals court in early November.
In August, the US Supreme Court ended the administration's pandemic-related federal moratorium on residential evictions.