Although the importance of nitrogen fixation in the Mediterranean Sea carbon and nitrogen fluxes is under debate, the diazotrophs responsible for this activity have still not been investigated in the open sea. In this study, we characterized the surface distribution and species richness of unicellular and filamentous diazotrophs by combining microscopic counts with size fractionated in situ hybridization (TSA-FISH) and 16S rDNA and nifH phylogenies. These genetic analyses were possible owing to the development of a new PCR protocol adapted for scarce microorganisms (1 cell ml-1). Diazotrophs community was dominated at 99.9% by picoplankton at a low concentration of 3.5 cell ml-1 including small (0.7-1.5 μm, UCYN-A) and large (2.5-3.2 μm, Crocosphaera watsonii) cells. NifH clone libraries were surprisingly dominated by Bradyrhizobium an α-proteobacteria closely related (94%) to a unique group of terrestrial rhizobia known to be able to fix nitrogen freely extra Plantae owning to their photosynthetic activity. We discuss the first discovery of C. watsonii and Bradyrhizobium in the Mediterranean Sea, as well as biotic and abiotic factors which may explain their distribution.