Protests against Babis have been going on for several weeks, as the billionaire PM has been involved in several investigations regarding fraud and conflict of interest, but he continues to reject a possible resignation.
Demonstrators argue that the new Minister of Justice, Marie Benesova, is too close to Mr Babis. "We'll see how many more people will still arrive", said Mikulas Minar, head of Million Moments for Democracy, the NGO organising the protest, as it got underway.
Police advise they're investigating accusations that Mr Babis hid his ownership of a farm and conference centre identified as "Stork Nest" (Capi hnizdo in Czech) so that it might most likely perhaps well qualify for an European Union subsidy created to encourage little companies.
"Demonstrations are part of the democratic system in the Czech Republic".
The crowd, carrying banners such as "Resign" and "We've Had Enough", was the largest in the country since the fall of communism there in 1989. Many families brought children to the rally, which was peaceful as were other recent protests against both Babis and his justice minister.
Mr Babis has repeatedly said he has no reason to resign.
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How has the prime minister reacted?.
The protesters of 2019 said they didn't come to overthrow the current political system but to defend it.
"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", said Rubas, 50. Politicians were not invited to speak at the rally which began at around 16.30 (1430 GMT).
Also, a preliminary report from the European Commission found Babis in conflict of interest because, despite having put his agriculture, media and chemical business empire in trusts, he has influence over EU funds they may receive.
Babis insists the audit, which said companies in the trust fund should not be eligible for European Union development subsidies, is wrong and this would be proven in the final conclusions, expected late this year or early in 2020.
The prime minister, whose ANO party leads in opinion polls, is also facing an opposition-led confidence vote in parliament in the coming days.