The Federation of Small Businesses (FSB) in Scotland said the report in yesterday's Herald, revealing Crawford Gillies has interests in several firms helped by Scottish Enterprise, will upset many of its 19,000 members.
Scottish Enterprise maintained it has robust systems in place to avoid a conflict of interest, and Mr Gillies has never had any involvement in investment decisions it has taken relating to companies in which he has a stake.
The Scottish Government also gave its backing to Mr Gillies yesterday.
But Colin Borland, the FSB's head of external affairs in Scotland, said: "Scottish small businesses that don't qualify for Scottish Enterprise support may be concerned when they read about the chairman's investments in organisations getting backing from the public purse.
"When public funds are under pressure, it's only right that those charged with their distribution face additional scrutiny."
The Scottish Trades Union Congress Deputy General Secretary, Dave Moxham, said there was no reason to believe impropriety has taken place.
However, he said: "I hope the chairman will give serious consideration to divesting himself of any relevant shareholdings.
"It is important that Scottish Enterprise now demonstrates that it has the appropriate governance arrangements in place to ensure that there is no risk of undermining the credibility of Scotland's main economic development body, and in particular that no board member stands to personally gain from the decisions Scottish Enterprise takes in its vital role in addressing the economic challenges Scotland faces."
The Scottish Green Party co-convenor, Patrick Harvie, intends to write to ministers asking for further information on the situation.
A spokeswoman for the Scottish Liberal Democrats added: "We are asking further questions about this in parliament.
"Ministers need to tell us if they are satisfied that adequate safeguards are in place within Scottish Enterprise to restrict the trading of equities by board members in companies assisted by Scottish Enterprise.
"People deserve assurances that there is transparency over the personal financial interests of the [chairman] in relation to companies which benefit from Scottish Enterprise funding."
A Scottish Government spokeswoman confirmed the Government continues to support Mr Gillies and were confident in the agency's procedures.
She said: "The Scottish Government values the continuing leadership Crawford Gillies gives to Scottish Enterprise and recognises his ongoing commitment to helping to deliver the Government's purpose of increasing sustainable economic growth.
"We are confident in the ability of Scottish Enterprise to build on its existing track record of helping Scottish companies to achieve their growth ambitions."