Provence-Alpes-Côte d'Azur was a target for Marine Le Pen's party.
But exit polls suggest their hotly-tipped candidate lost to the centre-right Republicans.
The election - which saw a potentially record low turnout of less than 30% - also brought disappointment for President Emmanuel Macron.
His centrist party, La République En Marche (LREM), also failed to win control of any region. It also performed badly in the first round, which was held last week.
It was the first time President Macron's party has taken part in regional elections, as it did not exist the last time they were held in 2015.
Ms Le Pen sought to win one of France's mainland regions for the first time in a bid to boost her presidential election hopes in 2022.
But early results from the second round suggest wins for traditional centre-right parties, and for the left.
In Provence, RN candidate Thierry Mariani lost to Republican candidate Renaud Muselier. With 75% of votes counted, Mr Muselier was more than 10% ahead of Mr Mariani.
"Tonight we have chosen the fate of a free region," Mr Muselier tweeted. Left-wing candidates withdrew from the race in the region to help the Republican defeat Mr Mariani.
Ms Le Pen accused her rivals of forming "unnatural alliances" to block her and her party from power.
"[They] did all they could to keep us out and prevent us from showing the French our capacity to lead a regional administration," she told supporters.
The Hauts-de-France region around Calais in the north had also been earmarked as a potential gain for Ms Le Pen's RN, but was won by conservative Xavier Bertrand.
"The far-right has been stopped in its tracks and we have pushed it back sharply," he told his supporters after the polls closed.
A big disappointment for Le Pen
President Macron's party was already sidelined in these elections after its poor showing in the first round.
Marine Le Pen's also scored badly last Sunday, but she at least had hopes of picking up one region - which would have been a first and a boost for her presidential bid.
It didn't happen - once again her voters stayed away from the polls - and overall these elections have been a big disappointment for the hard right.
The winners are the mainstream right whose candidates held on easily to the regions they already controlled. One of them the former minister Xavier Bertrand, whose fief is the northern Hauts-de-France region, is now openly a candidate for the presidency next year.