The Mediterranean Sea presents several biogeochemical anomalies compared to the global ocean. An unbalanced N budget, high nitrate/phosphate ratios in subsurface waters and low 15N/14N ratios in particulate and dissolved nitrogen suggest a significant occurrence of N2 fixation. This study presents, for the first time, a basin-wide overview of direct measurements of N2 fixation, with values in the North Atlantic for comparison, during late spring 2007. Very low N2 fixation rates (0.052 ± 0.031 nmols N l−1d−1) were observed in all sub-regions of the Mediterranean, in contrast to the higher values measured in the North Atlantic (0.300 ± 0.115 nmols N l−1d−1). Higher phosphorus (inorganic or organic) concentrations were not associated with higher N2 fixation rates. Low 15N/14N ratios in particulate organic nitrogen (from −2.10 to +4.11‰), associated with low N2 fixation rates, suggest that other N sources, such as atmospheric inputs, fuel the Mediterranean ecosystem.