Amounts paid to Intertek, which has a stock market worth of £4.3 billion, range from £600 to back a Middle East trade mission to £15,000 to help it find space for its Aberdeen operation.
Concern has grown since the quality and safety solutions firm said it had hired Scottish Enterprise chief executive Lena Wilson as a non-executive director.
Scottish Enterprise said Ms Wilson will take up this one-day-a-month role in a "personal capacity", in addition to her £200,000-a-year post at the economic development agency.
The move was endorsed by Scottish Enterprise chairman Crawford Gillies and Finance Secretary John Swinney.
Willie Rennie, leader of the Scottish Liberal Democrats, called for an investigation and said: "There is a clear conflict of interest, with Intertek having received financial support from Scottish Enterprise in the past."
Rhoda Grant, Scottish Labour's deputy finance spokeswoman, said: "John Swinney will have to answer some serious questions about why he agreed to this."
The Scottish Government said Mr Swinney was "aware of and supportive of the decision, but had no formal role in it".
A Scottish Enterprise spokes-man said grant contributions to Intertek since 2010 totalled about £31,000, and there had been none before that.
In 2011/12, grant assistance comprised about £6000 relating to a research and development project involving the University of Calgary, £2500 for strategy workshops in Aberdeen, £1500 towards presence at an overseas exhibition, and £600 to support a Middle East trade mission.
In the year to March 2011, assistance comprised a £15,000 grant to help identify appropriate space for Intertek "to retain the ... global headquarters of its upstream services in Aberdeen", a £2000 market launch grant, £700 for market development in the Middle East, £1000 for "activity in China" and £1500 for the R&D project involving the University of Calgary.
Dave Watson of Unison also raised concerns over "an obvious potential conflict of interest".
A spokesman for Scottish Enterprise said: "Lena has had no personal involvement with the company in either her present or previous roles in SE and 95% of Intertek's business is outside the UK. We've had a local relationship with Intertek's energy services business through our Aberdeen office since 2010.
"We have very clear policies in place to ensure no SE staff or board member is put in a position of conflict of interest. Our nature of operating, with private sector board members and extensive joint working with companies, means we are well versed in managing potential conflicts pro-actively."
Scottish Enterprise said on Monday the appointment was the first directorship of a publicly-quoted company taken up by a serving chief executive of the agency.
However, it has admitted Crawford Beveridge, who headed it in the 1990s, had been a non-executive director of ports operator Clydeport.