They broke into a secure compound in Yeoman Street, in the city centre, before spraying the Age UK vehicle on Tuesday night.
The vehicle, which ferries people to Clarence House in the city every day, was so badly covered in graffiti that it could not be used.
Silver paint blotted out nearly all the windows and black tags were also painted on the vehicle.
Age UK Leicester Shire and Rutland chief executive Tony Donovan said the damage was discovered at around 8am yesterday when staff arrived to take the ambulance on its round.
He said: "We will prosecute whoever has done this. It will have a very bad impact on the very vulnerable clients who use the ambulance every day.
"They see it is a lifeline to help them live independent lives. We had to take it off the road and bring in another vehicle from our fleet to take its place.
"We will also have to hire-in a replacement ambulance long-term while the repair work is carried out."
He said during disturbances in Leicester in the summer of 2011, one of its £45,000 mini-buses was badly damaged and clients at the time considered it to be an attack on them.
He said: "This graffiti attack will have a similar affect on those who use the ambulance."
He said the vehicle, which can carry 15 people, is used each day to bring people into the Clarence House day centre for a meal and to take part in activities designed to keep them motivated.
It picks them up from their homes in the morning and delivers them back again at the end of the day.
He added: "If it were not for this link, the people, who are very frail, would likely have to go into communal residential accommodation."
He said the cost to repair the damage was going to be thousands of pounds which would be paid out of funds that should be used to provide services.
He said: "The paint will have to be professionally removed and the vehicle resprayed to remove the black tags. We would urge anyone who saw what happened to contact the police."
A police spokeswoman said the incident in Yeoman Street was reported to them yesterday morning and officers were investigating what happened.
People who saw anything suspicious in the area are asked to contact the police on 101 or call Crimestoppers on 0800 555111.
Taken from: This is Leicestershire
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