In the latest round in a long-running dispute, organisers put turnout at 50,000, although police put the number at 15,000, albeit twice their estimate for a similar protest last month.
Sporadic violence marred a similar protest two years ago.
Protesters are demanding the airport plan be withdrawn and also the cancellation of expulsion orders handed to 11 families and four farmers living at the site.
They saw their appeals rejected last month.
The recent protests come amid a 15-year legal battle to block construction of a major new airport on marshland outside Nantes -- a project first mooted as far back as the 1960s.
Saturday's protest was the first since French President Francois Hollande said he would put the issue to a referendum.
Hollande agreed to the vote as a nod to green lawmakers who returned to government after storming out in protest over the airport plan in 2014.
"This demonstration is important to show our determination and show the politicians we outnumber them," said a spokesman for the marchers.
"We have a duty to preserve this land" for agricultural production he added.
Jose Bove, an ecologist and lawmaker in the European Parliament said "the only solution is for the president to declare this project not in the public interest."