JD Wetherspoon has said that 66 of its workers have tested positive for the coronavirus but maintains that visiting pubs is safe.
The company, which employs more than 41,000 people, said the vast majority of its pubs had recorded no positive tests for the virus.
There had been one or more cases among staff at 50 of its pubs.
Wetherspoon's boss Tim Martin dismissed claims by disease expert Professor Hugh Pennington that pubs are "dangerous".
He said: "The situation with regard to pubs has been widely misunderstood."
Aberdeen University's Prof Pennington said last month that pubs are "far, far more dangerous places to be" when discussing sending children back to school during the pandemic.
Since reopening on 4 July, the company said some 32 million people have visited its 861 open pubs.
Forty of its pubs have reported one worker testing positive for the coronavirus and six have disclosed two.
In addition, two pubs reported three staff testing positive and another two said four workers had.
A spokesman for Wetherspoon said the pubs have not been closed for a deep clean.
The company said it has invested £15m in hygiene and social distancing measures at its premises.
The spokesman said: "The advice Wetherspoon has received from the public health authorities is that employees should self-isolate if they come into close contact with someone who has tested positive. Close contact means within 2 metres for 15 minutes or more or 1 metre for 1 minute or more.
"Unless social distancing policies have not been observed, the health authorities, in our experience, do not normally advise closure."
He added that since the company's pubs reopened: "Staff are conducting regular surface cleaning and numerous hand sanitisers have been installed in each pub."
He said each premises is cleaned throughout trading and at the end of the day.
Wetherspoon said 28 of the affected employees had returned to work. A spokesman for the company said the workers, as well as those who worked in close proximity to them, self-isolated for 14 days and were paid in full.
The chain said signing up to the NHS track-and-trace system was mandatory in its premises.
The company's spokesman said there was no list of the pubs that had been affected. "Whenever there has been a situation we have dealt with the local press, public health and the council," he said.
Rule of six
Mr Martin argued that pubs and shops were safer than homes.
"It is much easier to inadvertently pass on the virus in someone's house, where people are more relaxed and less vigilant," he said.
From Monday, social gatherings of more than six people have been banned both indoors and outdoors in England and Scotland and indoors only in Wales.
It follows a rise in new coronavirus cases, with a further 3,330 positive cases recorded in the UK on Sunday.
Mr Martin said there had not been a rush of people coming to its pubs before the so-called "rule of six" was introduced and trade was 22.5% below the equivalent Saturday last year.
He added: "If pubs are closed, or restricted so much that they become unprofitable, a great deal of the strenuous effort of the hospitality industry's 3.2 million employees, currently engaged on upholding hygiene and social distancing standards, will be lost."