Unilever, pharmaceutical giant AstraZeneca and catering firm Compass Group were among leading FTSE- 100 businesses supporting a letter written by Downing Street’s “in” campaign.
But yesterday research by anti-Brussels campaigners showed many of the companies had received grants from the European Commission.
Many had also spent hundreds of thousands of pounds on lobbying in Brussels.
Around 200 business leaders employing 1.2 million people echoed the Prime Minister’s warning that an exit from the EU would put the British economy at risk.
Signatories included bosses of 36 FTSE-100 firms including Asda, BT, Marks & Spencer, Kingfisher and Vodaphone.
They said: “We believe leaving the EU would deter investment and threaten jobs. It would put the economy at risk.”
But the Vote Leave group found 34 of the firms received £93.5million in grants from the European Commission between 2007 and 2014.
AstraZeneca received £5.2million, defence firm BAE Systems £4.4million, Compass £29million, Unilever £1.6million and Vodafone £1million.
The same firms also spent up to £18million on lobbying in Brussels.
Vote Leave’s Robert Oxley said: “The EU is as bad for business as it is for our democracy and borders.
“It stifles competition, costs jobs and favours those with deep enough pockets to lobby behind closed doors.”
Campaigners said the majority of big British firms had refused to sign including Sainsbury’s, Tesco and Next, and banks Lloyds and Royal Bank of Scotland.
Richard Tice of the Leave. EU group, accused “bullying” Mr Cameron of using taxpayers’ money to “apply pressure” to companies.