Photos from the scene, in Watson St, show the ambulance on its side.
Paramedics assessed five people at the scene, including three who were taken to hospital.
“We sent paramedics and managers to the scene,” Ambulance Victoria said in a statement.
“We took an Ambulance Victoria uniformed staff member and a teenage boy to the Northern Hospital.
“A second Ambulance Victoria uniformed staff member was taken to the Royal Melbourne Hospital for further treatment.”
All three victims were in a stable condition.
Ambulance Victoria spokeswoman Lisa Hind said it was unclear with what the ambulance had collided with and officers were seeking more information.
An Ambulance Victoria helicopter was believed to be heading to the scene, near the Wallan police station.