Tui has cancelled all holidays to Florida until December following the introduction of new hygiene rules at Walt Disney World Resort (WDWR) such as mandatory face coverings.
The travel firm said it made the decision because the regulations would "significantly impact the holiday experience" for its customers.
WDWR, which includes four theme parks, will require visitors aged two and older to wear face coverings at all times except when eating or swimming once it reopens.
Temperature screenings may be required for entry to some locations, and the number of entry tickets will be limited.
Tui said in a statement: "As part of Tui's new holiday promise, the UK's biggest tour operator commits to only operating holidays where it's able to guarantee an enjoyable, relaxing and safe holiday without significant changes at their destination."
Customers are being offered a range of alternative options, including delaying their trip until next year or changing their destination - both with a booking incentive - or a full refund.
The company is to resume its holiday programme by serving eight destinations across Spain and Greece from July 11, as long as Government travel restrictions are eased.
WDWR is planning to partially reopen on July 15, but more than 11,000 people have signed a petition calling for a delay due to the recent increase in the number of coronavirus cases in Florida.
