An elephant and a hippopotamus had to be put down and a gorilla was feared to have drowned after the rising waters reached the Zoological Garden on the edge of the Czech capital.
The Indian elephant, called Kadir, was a large, older male and had been separated from the other elephants.
But he became trapped in his flooded enclosure, and zookeepers concluded the 35-year-old animal could not be rescued.
They decided to kill the elephant rather than let it drown.
Kadir has been a zoo favourite since 1968, and has even appeared in several Czech films.
His plight as the flood waters rose inspired Czechs to call the zoo with offers of money to help fund the rescue effort.
A hippopotamus also had to be put down after it escaped from its pen and became aggressive during the chaos.
Gorilla disappears
Hours after the two animals were put down, a gorilla vanished after flood waters submerged the pavilion where it was housed.
"We fear that the gorilla is no longer alive because the entire house where it used to live is under water," spokesman Vit Khale said.
He said the gorilla, a six-year-old male named Pong, remained missing, but that all other animals were safe.
Four other gorillas were sedated on Tuesday and transferred to another zoo in Dvur Kralove, about 100 kilometres (60 miles) to the east.
Sea lion escapes
On Wednesday, a sea lion escaped from the zoo and was spotted swimming in the rising waters of the Vltava River.
About 400 other animals were successfully moved to higher ground.
Workers used cranes to transport an elephant and two rhinoceroses.
Mr Khale said the zoo had received numerous calls from volunteers offering to help.
The Zoological Garden is situated on the low-lying and badly-flooded outskirts of the Czech capital, which has been hit by the worst flooding in more than a century.