Emergency services were alerted to the accident on the Melba Highway in Victoria's Yarra Ranges about 2.40pm on Thursday.
Images from the scene show the charter bus crashed on the side of the road after it is understood to have collided with a white car and blue Toyota.
The white car's driver died at the scene, police said, while multiple bus occupants were injured.
A teenage boy was flown to The Alfred hospital with serious injuries, while two teenage girls were taken for treatment with non-life-threatening injuries.
Paramedics also rushed a man in his 50s on the bus to Box Hill Hospital with serious injuries.
On-scene crews estimated the bus crashed with 18 people on board, including the driver.
The other passengers did not require emergency transport.
The driver and passenger of the third vehicle involved, a blue Toyota, sustained minor injuries.
An investigation into the incident is ongoing and Major Collision Investigation Unit detective remained at the scene on Thursday evening.
A specialist Air Ambulance Victoria helicopter crew and multiple Mobile Intensive Care Ambulance and Advanced Life Support paramedic crews assessed 21 people at the scene.
The bus was on scheduled V/Line coach service from Mansfield to Southern Cross.
The driver is an experienced driver with the charter company and was not seriously injured.Â
A section of the Melba Highway near Glenburn was closed to motorists over the afternoon.
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