Traffic was brought to a standstill after the collision near the Prince of Wales pub in Hailsham Road, Heathfield, at 6.15am on Wednesday.
Paramedics were on their way to an emergency call in the Heathfield area when they were involved in the collision with a black Honda Civic.
Police and fire crews rushed to the scene while another ambulance was dispatched to the original call-out.
The South East Coast Ambulance Service NHS Trust is now investigating the incident and police are appealing for witnesses.
Neighbours and nearby businesses on the busy road reported hearing a siren and then a loud bang.
Sally Philippou, owner of Tyrell’s Newsagents in Oxford Terrace, heard the accident from inside her store.
"I could hear the ambulance siren and then a crash. I heard a couple of crashes with no brakes. That’s what I thought was a bit strange because usually you hear a screech of brakes and then a thud," she said.
"I looked out and then I saw somebody get out of the ambulance, but he was sort of stunned, and then he went towards the other car.
"There were some cars whizzing up the road here so I was trying to slow them down.
"The police came and then a fire engine came and then more police."
Louisa Hill, of Hailsham Road, was woken up by the crash.
She said: "We heard the sirens and then heard a big bang, sort of all together really.
"It was very dark. I could just see out the window that someone was in the black car, obviously not moving, and then the fire engine came and we heard them start up their cutting equipment."
Crews from Heathfield and Mayfield fire stations were sent to the scene and had to free the driver of the Honda by removing the car’s right-hand door.
The 29-year-old car driver, from Heathfield, and the front seat passenger in the ambulance, a 24-year-old man, were both taken by another emergency vehicle to Eastbourne District General Hospital with minor injuries.
The ambulance driver, a 47-year-old man, was uninjured.
Witnesses said the road was closed until 9am, disrupting rush-hour traffic and the school run.
