HM Revenue and Customs have been urged to look into the possibility that Tony Blair and other ministers enjoyed tax benefits by using the Royal Flight.
Mr Blair used helicopters to get around during the election
Conservatives want the Revenue to investigate whether flights count as "benefits in kind" for tax purposes.
Mr Blair is also under fire for using an RAF helicopter to visit the West Midlands ahead of last year's election.
Downing Street insisted the flight by the prime minister and Chancellor Gordon Brown was legitimate.
The two men flew to MG Rover's Longbridge plant, following the firm's collapse, as part of a bid to save it.
But the Conservatives said making the flight at the taxpayers' expense at such a politically sensitive time appeared "inappropriate", particularly as Labour was defending two key marginal seats nearby.
The flight was made 10 days into last year's general election campaign.
Number 10 say the visit was government business to secure agreement on a £150m support package for workers who had lost their jobs in the collapse.
Shadow transport secretary Chris Grayling said the visit had clearly been a "high profile political event" in the middle of the election campaign.
"Whatever the detail of the rules, surely he must realise that most sensible people would say this really looks inappropriate," he said.
Mr Grayling also wants to know if Mr Blair is liable for extra tax for his family holiday flights, for example a £31,000 trip to Sharm el-Sheikh in 2004 even if he used the Queen's Flight for security reasons.
The Conservatives plan to table a series of parliamentary questions on the issue next week.
The aircraft involved was part of 32 (The Royal) Squadron - the successor to the former Queen's Flight - which provides transport to members of the Royal Family and senior government ministers.
Earlier this week official figures were released showing that Mr Blair had made 622 flights using RAF aircraft since becoming prime minister - including some for family holidays - at a total cost of £1.22m.
But the Tories say that these figures do not cover other costs such as depreciation on the value of aircraft.
Mr Blair's director of communications Dave Hill, meanwhile, has written to the Times defending his boss.
"Tony Blair is the first prime minister in recent times not to use RAF aircraft for family holidays. John Major and Margaret Thatcher both did so and paid the cost of an equivalent commercial flight for their families," he stated.
"Mr Blair followed this custom until 2000 when he decided the practice was difficult to justify. Since then he has used commercial flights for holidays whenever possible."
The figures - which were made available under the Freedom of Information Act - only went up to the end of February last year and did not cover the election campaign which began in April.
Under strict Whitehall rules, ministers can only use RAF flights on government business and not for political engagements. A Downing Street spokeswoman said the Longbridge visit had been "straightforward government business".
Mr Blair and Mr Brown had made the trip accompanied by civil servants.
"It is nonsense to suggest that this was not a government issue when you were talking about £150m of government money," she said.
Meanwhile the Tories want to know why Margaret Beckett, in her role as environment secretary, had taken RAF flights to Brussels when she and her officials could have taken a train for meetings with fellow EU ministers.
"No way is it cheaper to charter a plane than to take Eurostar," said a Conservative spokesman.