The policy means the company will no longer hire people who aren't fully vaccinated, while current employees will have 60 days to get their jabs sorted.
It comes after Facebook, Google and Netflix announced similar policies.
In a statement, Disney said: "At The Walt Disney Company, the safety and well-being of our employees during the pandemic has been and continues to be a top priority.
"Toward that end, and based on the latest recommendations of scientists, health officials and our own medical professionals that the Covid-19 vaccine provides the best protection against severe infection, we are requiring that all salaried and non-union hourly employees in the US working at any of our sites be fully vaccinated.
"Employees who aren't already vaccinated and are working on-site will have 60 days from today to complete their protocols and any employees still working from home will need to provide verification of vaccination prior to their return, with certain limited exceptions."
The statement continued: "We have also begun conversations around this topic with the unions representing our employees under collective bargaining agreements.
"In addition, all new hires will be required to be fully vaccinated before beginning employment.
"Vaccines are the best tool we all have to help control this global pandemic and protect our employees."
US President Joe Biden recently announced sweeping new pandemic requirements aimed at boosting vaccination rates for millions of federal workers and contractors, as he lamented the 'American tragedy' of deaths among the unvaccinated.
Federal workers will be required to sign forms attesting they have been vaccinated against Covid-19, or else comply with new rules on mandatory masking, weekly testing, distancing and more.
The strict new guidelines are aimed at increasing sluggish vaccination rates among the huge number of Americans who draw federal paycheques.
They are also designed to set an example for private employers around the country.
Biden said at the White House: "Right now, too many people are dying or watching someone they love die and say, 'If I'd just got the vaccine.'
"This is an American tragedy.
"People are dying who don't have to die."
Seemingly fed up with persistent vaccine resistance among many Americans, he also delivered a sharp rebuke to those who have yet to get their jabs, saying 'they get sick and fill up our hospitals'.
He said: "If in fact you are unvaccinated, you present a problem to yourself, to your family and those with whom you work."
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