Andrew Lloyd Webber said the "blunt instrument that is the government's isolation guidance" has forced him to halt performances of his new musical, Cinderella, in London.
The show, which has been written by Oscar-winning Emerald Fennell, was due to have a "Freedom Day" performance on Monday evening after the government lifted its Covid restrictions.
It is understood the show will be closed for at least the rest of the week and the weekend.
Cinderella was forced to close earlier, on Saturday, after a positive test in the team.
"Freedom Day has turned into closure day," the theatre magnate said.
"At Cinderella, from the outset, we have employed a rigorous testing system for all the cast and backstage crew before they begin work.
"On Saturday, as part of this process, we identified one positive case in a member of our cast who has a cameo role in the show.
"As a precautionary measure, we cancelled two shows ... while we carried out further tests on everyone backstage, which were negative.
"Any of those who were identified as a close contact of the positive case were given additional PCR tests. These tests, too, were negative.
"This morning we carried out additional tests on those due to perform tonight [Monday]. Every one of them was negative.
"Despite this, the impossible conditions created by the blunt instrument that is the government's isolation guidance mean we cannot continue.
"We have been forced into a devastating decision which will affect the lives and livelihoods of hundreds of people and disappoint the thousands who have booked to see the show."
The musical's lead actress, Carrie Hope Fletcher, shared her sadness at the show's closure, writing on Instagram: "So sorry to everyone who had tickets to the show, however many performances won't be going ahead.
"From the bottom of my heart, I'm so, so sorry."
The show opened last month at 50 per cent capacity after the government extended the Covid-19 restrictions.
Lloyd Webber was offered the chance to take part in the government's Events Research Programme, but he rejected it.
He has been critical of the government in recent months, saying he would be prepared to be arrested to fully reopen his theatres on June 21 if the easing of lockdown restrictions were delayed.
But Lloyd Webber later admitted that it would leave his cast and crew with hefty fines.