Simon Walker, director general of the Institute of Directors (IoD), said that Britain could experience years of job losses, higher prices and slower economic growth if it voted to leave.
A post-EU Britain could see "up to 10 years of relative uncertainty -- and that is a profound disincentive for many businesses," he said.
Walker, whose organisation represents around 35,000 executives, mostly at small and medium businesses in all sectors of British industry, said the majority of the business community favoured staying in the union.
His comments came after 197 UK business leaders signed a letter to The Times on Tuesday backing a vote to stay.
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Walker said small and medium-sized businesses would be among the most vulnerable if Britain left because they tend to export to other European countries rather than markets such as China and India.
He added that London's tech scene, which hosts nearly 40,000 businesses employing over 250,000 people, would suffer if European start-ups could no longer move there easily.
A poll of members of the Federation of Small Businesses in September 2015 showed that 47 percent would vote to remain in the union, against just under 41 percent who would vote to leave.
But the arguments for Brexit tend to focus on sovereignty and immigration, rather than a business logic, Walker said.
"If the people of this country take measures that end up cutting jobs, depressing the value of the pound, limiting economic growth, that is their absolute democratic right," he said.