The First Minister has been urged by Labour MSP James Kelly to look into whether there was a breach of the Scottish Ministerial Code after the event in May.
It comes after ministerial journeys were revealed in official travel logs published online earlier this week.
The logs show five SNP cabinet secretaries and four ministers used seven government cars to travel from Perth Concert Hall to Edinburgh on May 30.
The ministerial code states that “official transport should not normally be used for travel arrangements arising from party or private business”.
But the government claims there was no wrongdoing as the ministers were travelling to carry out official duties.
Mr Kelly said he had written to Nicola Sturgeon asking her to “investigate rules breach by ministers taking government cars to the SNP manifesto launch”.
“These are serious accusations that must be immediately investigated,” he said.
“The ministerial code is a vital part of our democracy and is essential to retaining public trust in politicians.
“The public and parliament deserves to know how these trips came to take place, what the justification for them is and whether such journeys will happen again.
“Anything less than a full investigation would be a breach of trust and leave SNP ministers looking even further out of touch than they do already.”
Tory MSP Miles Briggs claimed the revelation was the “kind of hypocrisy and abuse the SNP is becoming renowned for”.
“The public will be deeply uncomfortable at the idea of eight ministers swanning out of such a party political event and straight into a taxpayer-funded car,” he said.
“And they obviously took their lead from Nicola Sturgeon a month earlier, who may have enjoyed posing on a Yes-themed motorbike for a publicity stunt. But what the cameras didn’t pick up was the taxpayer-funded fancy car waiting round the corner.
“Anyone can see just how political events like a manifesto launch or photo call are, and the ministerial code clearly states this kind of thing would be a breach.
“SNP ministers seem to have forgotten that these cars are paid for by the public, and they risk not being taken seriously when they complain about limited resources available.”
Details of the First Minister’s car journeys in May and June are not known as the Government no longer makes these public for “security reasons”.
A Scottish Government spokesperson said: “The Government car service is available for the use of ministers and cabinet secretaries in travel to and from Government business.
“On this occasion, ministers and cabinet secretaries were picked up and travelled to a variety of ministerial engagements.
“We continually look for ways to minimise the use of cars for official journeys, for example by car sharing as happened on this occasion.
“Where practical to do so, ministers also use public transport or walk to their engagements.
“This is not always practical if it is necessary for them to carry out sensitive government business during their journey.”