Bird flu was reported last Friday at the farm located 150 km (90 miles) west from Prague, which can house up to 750,000 chickens, after an increase of deaths in one of three halls there.
The Czech Republic's State Veterinary Administration (SVS) said on Tuesday that since checks revealed the infection only in one of the halls, the majority of the flock could be spared.
"The whole (population of the) hall will have to be culled," said Petr Majer, SVS spokesman.
The infections come as countries try to limit the spread. Since November, Czech authorities have required poultry farms to keep flocks indoors.
On Tuesday, another case of bird flu was reported at a farm 68 km south of Prague, where 12,000 chickens and 1,000 turkeys will be culled, CTK news agency reported.
Bird flu is pressuring already high food prices and triggering trade restrictions from countries that import poultry.
The virus can be transmitted to humans in contact with poultry, but experts say the health risk to humans is low.