The river reached its highest point so far on Wednesday afternoon and authorities said the coming hours would be a critical test of the hastily constructed flood defences.
Water levels, which had been rising at a rate of about 10 to 15 centimetres per hour, are now said to be increasing more slowly.
The rain has now stopped, but correspondents say the situation remains very confused and the authorities are at something of a loss to predict the next stage of the disaster.
Floods have swept across much of the country, killing nine people and forcing 200,000 nationwide to be evacuated.
The government has deployed 4,000 soldiers and 15,000 police, firemen and volunteers are participating in the emergency efforts.
Czech Foreign Minister Cyril Svoboda has said his government would welcome offers of aid from abroad to help the devastation to the country's already fragile economy.
Floods have devastated swathes of other central and East European countries, killing about 90 people:
Fighting a phenomenon
On Wednesday morning, water swept into the basement of Prague's National Theatre.
River water seeped through sandbags protecting the residential Smichov district and elsewhere water has been bubbling up from the sewers.
"It is the beginning of the worst moment," said river authority spokesman Vaclav Baca. "All of the flood barriers are at their maximum level".
But, according to Mr Baca, the situation is set to improve, as less water flows into the Vltava from the Orlicka reservoir and the levels of the Berounka tributary river decrease.
According to water experts, about 5% to 8% of the city has been inundated, making it the worst flooding in 200 years.
Prague Mayor Igor Nemec said: "We're fighting a phenomenon".
If the river does burst the barriers, it will inundate the spectacular Old Town Square and several historic monasteries.
At Prague Zoo, about 400 animals were moved to higher ground.
Zookeepers put down a 35-year-old elephant and a hippopotamus. A gorilla is feared drowned.
In the early hours of Wednesday morning, new areas of the city were evacuated, as earlier hopes that the floods would abate were dashed.
The city authorities have asked people to take enough food, drink and clothing with them for four to five days.
Nationwide crisis
The floods have already inundated southern parts of the Czech Republic.
Road and rail lines have been cut, and towns and cities swamped.
There are fears of an ecological disaster in the South Bohemian town of Strakonice, where oil from a factory has leaked into the water supply.
The towns of Plzen and Ceske Budejovice - famous centres of Czech beer production - are beginning to recover after being affected earlier in the week.
But the important industrial towns of Usti nad Labem and Mlada Boleslav expect the worst is still to come.
Evacuation orders have been issued for 3,000 people in parts of Luznice na Taborsku and overnight more than 5,000 people were evacuated from in and around Znojmo na Dyji.