Professor Sir Timothy O'Shea, principal of Edinburgh University, said a "principal anxiety" over the Post-16 Education (Scotland) Bill was the prospect of intervention by ministers.
Professor Seona Reid, director of Glasgow School of Art, also attacked the proposed legislation, saying it could be "misused".
The attacks came after Michael Russell, the Education Secretary, published the bill last year.
The legislation lets ministers set priorities for universities in return for their public funding – including ensuring institutions comply with new rules on governance and widening access.
It also gives the Scottish Funding Council (SFC), which administers public money on behalf of the Government, powers to review the provision of courses.
Giving evidence to the Scottish Parliament's Education Committee, which is scrutinising the legislation, Sir Timothy said the spirit of the bill was welcome, but highlighted a "principal anxiety".
"We are very supportive of the whole bill in terms of intentions with regards to widening participation, greater efficiency of the sector and greater accountability," he said. "At the same time, we are very aware that the Scottish universities are seen as being particularly successful, and the outside commentators relate that success to the responsible autonomy that we discharge.
"We are anxious that there may inadvertently be a potential reduction in responsible autonomy and that some future administration might be in a position to intervene in a way that would be unhelpful to the success of universities.
"Our anxiety is that if a future government were given the apparatus whereby it could intervene in our management or governance structures then it might choose to do so."
Ms Reid added: "The issue of a code of governance is something that all universities, of whatever size or nature, support absolutely wholeheartedly.
"But to enshrine it in legislation risks the possibility that for future administrations it could be misused to apply a uniformity of governance model that would be inappropriate to a diverse sector."
However, Labour learning and skills spokesman Neil Findlay challenged the concerns, suggesting conditions may have to be imposed to address the shortcomings of institutions that have "failed miserably" to widen access to their courses.
"I would suggest part of the reason why the Government is seeking to legislate is because of the approach that you are presenting to us today," he said.
"They feel that things haven't gone far enough, therefore they feel as though they are going to have to legislate."
Later, committee convener Stewart Maxwell quoted Tracey Slaven, from the Government's Bill team, who told the committee last week they are listening to the sector's concerns about "unintended consequences" of the bill.
Mr Maxwell said: "It sounds like they are certainly reconsidering their position. Is that your understanding?"
Sir Timothy said: "I met with Tracey Slaven yesterday and that was my exact understanding."
A Scottish Government spokesman said: "The Government is of course listening to all points of view during this process of pre-legislative scrutiny."