An estimated 250,000 people joined the rally demanding the resignation of their prime minister, the culmination of a series of demonstrations in recent months.
The protesters say Andrej Babis, the populist billionaire leader, is a threat to democracy, including the independence of the country’s legal system.
Mr Babis has faced investigations over alleged fraud and conflicts of interest, claims he vehemently denies.
Prosecutors are considering whether to charge the prime minister with fraud involving European Union funds.
Protesters carried banners saying “Resign”, “We’ve had enough” and “We won’t give up democracy”.
Many families took children to the rally, which was peaceful, as were other recent protests.
The demonstrators also are demanding the resignation of Marie Benesova, Mr Babis’ new justice minister, claiming she might influence legal proceedings. That possibility has also been vigourously denied.
Mr Babis has said people have the right to protest but has firmly refused to step down.
His populist ANO movement remains the most popular party, although its support has dipped slightly in two months to 27.5 per cent, according to the most recent poll.
He also has enough backing in parliament, where a no-confidence vote planned for Wednesday is likely to fail.
Police proposed in April that Mr Babis, a billionaire businessman-turned-politician sometimes likened to Donald Trump, should be formally charged for fraud in tapping an EU subsidy a decade ago to build a hotel and conference centre.
“Our country has many problems and the government is not solving them. It is not solving them because the only worry of the prime minister is how to untangle himself from his personal problems,” said Mikulas Minar, a 26-year-old student who set up the Million Moments for Democracy, a group that organised the protests.
“It is unacceptable for our prime minister to be a person under criminal investigation,” he told the crowd from a giant stage.
Filip Rubas, 50, who joined protests in 1989 against the then-communist regime, said: “We think our leaders need to be reminded very strongly that they do not own our country, that they are not above the law (or constitution) and that there are still enough caring people who are not brainwashed by hateful propaganda.”
Leaked preliminary results of an audit by the European Commission said that Mr Babis, 64, was in conflict of interest as the beneficiary of trust funds to which he had transferred his chemicals, farming, media and food business, valued by Forbes at $3.7 billion (£2.9 billion).
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