Biological nitrogen fixation by diazotrophic bacteria in seagrass rhizosphere and leaf epiphytic community is an important source of nitrogen required for plant growth. However, the presence of endophytic diazotrophs remains unclear in seagrass tissues. Here, we assess the presence, diversity and taxonomy of nitrogen-fixing bacteria within surface-sterilized roots of Posidonia oceanica. Moreover, we analyze the nitrogen isotopic signature of seagrass tissues in order to notice atmospheric nitrogen fixation. We detected nitrogen-fixing bacteria by nifH gene amplification in 13 out of the 78 roots sampled, corresponding to 9 locations out of 26 meadows. We detected two different types of bacterial nifH sequences associated with P. oceanica roots, which were closely related to sequences previously isolated from the rhizosphere of a salt marsh cord grass and a putative anaerobe. Nitrogen content of seagrass tissues showed low isotopic signatures in all the sampled meadows, pointing out the atmospheric origin of the assimilated nitrogen by seagrasses. However, this was not related with the presence of endophytic nitrogen fixers, suggesting the nitrogen fixation occurring in rhizosphere and in the epiphytic community could be an important source of nitrogen for P. oceanica. The low diversity of nitrogen-fixing bacteria reported here suggests species-specific relationships between diazotrophs and P. oceanica, revealing possible symbiotic interactions that could play a major role in nitrogen acquisition by seagrasses in oligotrophic environments where they form lush meadows.