"I can confirm we could not help two people," Prague ambulance spokeswoman Jana Postova told AFP, declining to give details about the victims.
"We are treating seven people, of them three are in a serious condition. Two needed lung ventilation," she said, adding about 40 people had to be evacuated from the four-star Eurostars David hotel in the Czech capital's centre.
Martin Kavka, spokesman for the Prague fire brigade, told AFP the blaze was reported after 1700 GMT and that firefighters had it under control about two hours later.