Authorities argue that the new airport will provide a major boost to tourism in western France, but environmental campaigners have fiercely opposed the plans to build it on protected swampland just outside Nantes.
Thousands of protesters, some on foot, others on bikes or driving tractors blocked roads in the area demanding the cancellation of expulsion orders handed to 11 families and four farmers living at the site.
Organisers said 20,000 people took part, but police put the number at 7,200.
Some agricultural organisations threatened to maintain an indefinite blockade of one of the main bridges over the Loire river, but police intervened with tear gas in the night and by midnight the route had been cleared.
Protesters have been engaged in a 15-year legal battle to block construction of the new airport, with Saturday's demonstration the biggest gathering in two years.
Demonstrators caused major disruption to traffic on the Nantes ring road and also blocked access to the city's international airport, Nantes Atlantique.
But unlike the protests of February 2014, which gathered over 20,000 people and deteriorated into clashes with the police, Saturday's rally took place in a peaceful atmosphere.
Agricultural organisations using megaphones had announced that they would keep one of the main crossing points over the Loire, the Chevire bridge, blocked as long as French President Francois Hollande failed to stop the expulsion orders.
To begin with, between 80 and 100 tractors would block access to the bridge on Saturday night, Vincent Delabouglisse, of the anti-airport organisation COPAIN44, told AFP.
The protest was held two months after an announcement by regional authorities that the massive construction project, which has been on hold for nearly three years, would resume in 2016 in a move backed by the courts.
The project involves transferring Nantes Atlantique airport to a 1,650-hectare (4,000-acre) site of protected swampland just outside the city, which developers say will provide a major boost to tourism in western France.
Approved in 2008, the 580-million-euro ($747 million) project had been due to start in 2014 but has been repeatedly delayed due to fierce opposition by environmental protesters.