Five French sprinters were battling for the two men's places in the event at the London Olympics: Bernard, Amaury Leveaux, Yannick Agnel, Fabien Gilot and Gregory Mallet.
All five had dipped below the 49-second barrier in the semi-finals on Wednesday, but a day later it was Agnel who led Gilot home in a personal best of 48.02, the best time of the year for a French swimmer.
Gilot was second in 48.38 as Bernard underperformed with a time of 48.97 that was only good enough for fifth place.
"It was a quick swim and I didn't swim fast enough," said Bernard, who also failed to make the 100m at the world championships in Shanghai last year.
"There is disappointment, for sure, because I felt I could do a little better. But I didn't do it."
Despite finishing only in fifth, it is believed Bernard has a good chance of being retained for the men's 4x100m relay team.
Agnel has also secured his spot for the 200m freestyle after winning the national title here, meaning he will line up in London as France's big hope for the men's sprint events.
However the Frenchman's winning time is still a way behind the blistering times set by Australian pair James Magnussen (47.10) and James Roberts (47.63).
"I'm not going to get carried away because there is a lot of work to do before I can reach the level of the Australians," said Agnel.
In third place was Clement Lefert (48.64) with Leveaux fourth in 48.69.