32% of small and medium businesses said the EU hinders businesses like theirs, while 25% said it helped them and 40% said it made no difference
The Vote Leave group has published a list of 250 business leaders who are backing the campaign for Britain to leave the EU.
It includes former HSBC chief executive Michael Geoghegan, Tim Martin, chairman of JD Wetherspoons and Carphone Warehouse founder David Ross.
Vote Leave has also announced its Business Council will be chaired by John Longworth, who resigned as director general of the British Chambers of Commerce (BCC) earlier this month.
It followed his suspension for saying the UK's long-term prospects could be "brighter" outside the EU despite the BCC maintaining a neutral position.
Mr Longworth said he was "delighted" to be joining the Brexit campaign, adding: "This is the most important political debate of a generation.
"Many firms struggle with relentless interference from the EU and rules that are stacked in the favour of a select number of businesses.
"If we Vote Leave, liberated from the shackles of EU membership, jobs will be safer… and we can look forward to faster growth and greater prosperity in the future."
His comments were echoed by Vote Leave's chief executive Matthew Elliott.
"Brussels hinders smaller businesses, particularly those firms who can’t afford to lobby Brussels to curry favour," he said.
"Jobs, wages and our economy will thrive when we take back control and Vote Leave."
A survey of small and medium-sized firms commissioned by Vote Leave found that 32% said the EU hinders businesses like theirs, while 25% said it helped them and 40% said it made no difference.
The YouGov study was dismissed as one-sided by Lucy Thomas, deputy director of Britain Stronger in Europe.
She said: "Survey after survey has shown that businesses of all sizes and from all sectors overwhelmingly back Britain remaining in the EU.
"It is telling that Vote Leave's poll doesn't even ask the most important question about whether Britain should remain in the EU because they know most businesses - large and small - disagree with them.
"Instead they resort to leading questions about regulation."
The referendum, which follows a new deal on EU membership negotiated by David Cameron, will be held on 23 June.
:: YouGov surveyed 1,002 SME decision-makers online between 14 and 21 March.