The fire started on the fourth floor of an eight-story building in a shopping and entertainment area of Kitashinchi, Osaka city fire department official Akira Kishimoto said.
Twenty-eight people were affected, 27 of whom were found in a state of cardiac arrest, Kishimoto said. So far 23 people have been taken to nearby hospitals, he said.
The building houses an internal medicine clinic, an English language school and other businesses, NHK national television said.
The cause of the fire and other details were not immediately known. NHK said Osaka police are investigating the case as a possible arson.
People on other floors of the building were believed to have been evacuated, Kishimoto said.
NHK footage showed dozens of fire engines and police vehicles on the street near the building, with onlookers watching the development from across the street.
In all, 70 fire engines were mobilized to fight the fire, which was mostly extinguished within about 30 minutes of an emergency call, officials said.