The leader of the anti-immigration FN, Marine Le Pen, lost out to the right-wing opposition in the northern Nord-Pas-de-Calais Picardie region after the ruling Socialists pulled out of the race before the second round.
Her 26-year-old niece Marion Marechal-Le Pen was also defeated by the right-wing grouping in the southern region that includes the glitzy resorts of the Cote d'Azur, despite dominating the first round last week.
The FN had topped the vote in six of 13 regions on December 6, but once again suffered in the second round of an election as voters rushed to support mainstream parties at the final hurdle.
With turnout high on Friday at around 59 per cent, the right-wing grouping appeared to have won at least five of the regions.
The election was held under tight security following the November 13 attacks in Paris in which 130 people were killed.
Despite the FN failing to grab its first-ever region, Marine Le Pen will still use her party's performance as a springboard for her bid for the 2017 presidential election.
The FN has topped European and local polls over the past two years, bolstering Le Pen's claim that it is now "the first party of France". It controls around 11 towns across the country.
But today showed once again that the party struggles in the deciding round as mainstream voters gang up to keep it from power.
Early estimates showed Marine Le Pen scored around 42 per cent to nearly 58 per cent for her right-wing rival Xavier Bertrand in the economically depressed north.
Her niece scored around 45 per cent to nearly 55 per cent for her right-wing rival in the FN's traditional stronghold in the south.
The FN argues that the political manoeuvring by the main two political parties shows that they are two sides of the same coin and the far-right offers the only real political alternative.
"Frankly, I'm voting against the FN in the interests of my family," said Issa Kouyate, a 59-year-old voter of Senegalese origin, as he went to cast his ballot in the southern city of Marseille, where a high proportion of citizens are of immigrant background.
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