Some 198 business leaders including Roger Carr, chairman of BAE Systems, BP CEO Bob Dudley and Ron Dennis, chief of F1 team McLaren, wrote a joint letter published in the Times, backing Prime Minister David Cameron's deal to reform the EU, AFP reported.
"Following the prime minister's renegotiation, we believe that Britain is better off staying in a reformed European Union," they wrote, adding Cameron had secured important commitments to improve competitiveness within the bloc.
"We believe that leaving the EU would deter investment, threaten jobs and put the economy at risk," wrote the business chiefs, who between them employ around 1.2 million people.
"Britain will be stronger, safer and better off remaining a member of the EU," it concluded.
The letter comes as a timely boost for Cameron, who was rocked Sunday by the decision of charismatic London mayor Boris Johnson to back a "Brexit".
Chairmen or chief executives of 36 companies from key share index FTSE 100, including national giants such as Asda, BT, Marks & Spencer and Vodafone, all signed the letter.
However, critics pointed out that many large employers such as Tesco, RBS, Barclays and Sainsburys had not signed the letter and accused Cameron of "bullying" businesses into supporting his position.
"The truth is that despite the bullying of a prime minister who has no real business experience, it is other normal commercial factors which will determine the continued success of British businesses to invest and grow," said Richard Tice, co-founder of pro-Brexit group Leave.EU.
"Brexit will reduce unnecessary regulatory burdens and cost on business, which can be used to invest in more jobs, not less," he added.
Despite Cameron's deal, bookmakers have slightly reduced the odds on Britain voting to leave while sterling sank to its lowest value against the dollar since 2009.
Rating agency Moody's threatened to downgrade Britain's AA1 rating to "negative outlook" if it voted to leave the EU.