The UK's car industry, more leading businessmen, and the chairman of football's Premier League have backed the campaign to stay in the EU.
In a joint statement, the car industry's trade body and company executives warned that leaving would increase costs and threaten jobs.
Sir Richard Branson, and the chief executive drinks giant Diageo, Ivan Menezes, have also supported Remain.
Vote Leave said UK-made cars were exported worldwide, not just to the EU.
The group also dismissed the intervention by the two businessmen, claiming they have already voiced pro-EU support and the latest comments were no surprise.
The Society of Motor Manufacturers and Traders' (SMMT) chief executive Mike Hawes said a vote to leave the EU would "jeopardise" the industry's continued success.
"Remaining will allow the UK to retain the influence on which the unique and successful UK automotive sector depends," he said.
Support for a Remain vote in Thursday's referendum also came from senior automotive executives of an industry which the SMMT said supports 800,000 jobs and contributes £15.5bn annually to the UK economy.
Directors at Toyota UK, Vauxhall, Jaguar Land Rover and BMW, as well as from component makers GKN and Magal Engineering, voiced their support.
Rory Harvey, chairman of Vauxhall, said: "We are part of a fully integrated European company where we benefit from the free movement of goods and people. We believe not to be part of the EU would be undesirable for our business and the sector as a whole."
'Wrong then, wrong now'Vote Leave issued a detailed rebuttal of the industry's claims, citing several comments by car executives who said Brexit would not harm jobs and investments.
The group's chief executive, Matthew Elliott, said: "We know that British car exports are increasingly going to the rest of the world, not the EU. As such, a vote to leave could provide a boost to the industry as we would be free to sign free trade agreements with emerging markets - something we are currently forbidden from doing by the EU."
He also pointed out that many car company executives had urged Britain to join the euro. "They were wrong then and they are wrong now," he said.
Meanwhile, Diageo's chief executive has written to the company's 4,773 UK employees, telling them that it would be "better for the UK, better for Diageo and better for the Scotch whisky industry that we remain in".
Mr Menezes said Diageo benefitted from ease of access to the European single market, as well as trade deals that the EU has negotiated with the rest of the world.
'Saddened'"The EU has so far concluded, or is negotiating, over 50 of these global agreements, many of which provide significant commercial benefits for Diageo," Mr Menezes said in his letter. Negotiating new deals after a Brexit could take years, he said.
Sir Richard Branson also voiced support for Remain on Monday in an open letter in which the Virgin Group boss said Brexit would be "devastating" for the long-term prosperity of the UK.
He recalled "how difficult it was" for businesses to operate effectively before the EU, adding he was "saddened" at the prospect of returning to those days.
He wrote: "Although I've been living in the British Virgin Islands for some time now, I have never stopped caring passionately about the UK and its great people. I am one of the few business people who can remember how difficult it was before the EU was formed."
Vote Leave dismissed the comments from Sir Richard and Mr Menezes as "no surprise" and "not new". A spokesman said: "These are people who support Remain saying again that they support Remain."
Meanwhile, Richard Scudamore, executive chairman of the Premier League, pledged his support to remain in the EU, and said the 20 clubs in the top tier also wanted to remain.
Grassroots sportHe told BBC 5 live that leaving would be "incongruous" in the context of the league's commitment to "openness".
"There is an openness about the Premier League which I think it would be completely incongruous if we were to take the opposite position," he said.
But the Leave camp have criticised Mr Scudamore's assessment, saying Brexit could shore up more funds to be invested in grassroots sports.
Vote Leave's Robert Oxley said: "EU rules hurt both our ability to develop home grown talent and restrict access to the global talent pool.
"That's the worst of both worlds for football fans and the teams they support. If we take back control, we can spend the £350m we send to Brussels each week on our priorities, like grassroots sport."
UndecidedAs Thursday's Referendum approaches, executives have recently stepped up support for the Leave and Remain camps.
On Sunday, the chief executive of energy giant EDF Energy, Vincent de Rivaz, wrote to his UK staff outlining what he says are the benefits of continued EU membership.
Earlier this month Lord Bamford, chairman of JCB, one of Britain's most successful manufacturers, wrote to his company's 6,500 employees in the UK to explain why he favours a vote to leave the European Union.
Last weekend, entrepreneur Sir James Dyson told the Daily Telegraph Britain would gain more from leaving the EU than it would lose.
Last week, BT bosses and union leaders came out in favour of the Remain camp.
Big business and financial firms have generally been in favour of staying in the EU, although surveys suggest that small businesses are evenly split.
Last week, a poll by the Federation of Small Businesses suggested that 42% of members "could still be swayed on how to vote" with 52% "saying they didn't feel they had the information they needed".