The western tropical South Pacific (WTSP) Ocean has been recognized as a global hotspot of dinitrogen (N2) fixation. Here, as in other marine environments across the oceans, N2 fixation studies have focused in the sunlit layer. However, studies have confirmed the importance of aphotic N2 fixation activity, although until now only one had been performed in the WTSP. In order to increase our knowledge of aphotic N2 fixation in the WTSP, here we measure N2 fixation rates and identify diazotrophic phylotypes in the mesopelagic layer along a transect spanning from New Caledonia to French Polynesia. Because non-cyanobacterial diazotrophs presumably need external dissolved organic matter (DOM) sources for their nutrition, we also identified DOM compounds using Fourier Transform Ion Cyclotron Mass Spectrometry (FTICRMS). N2 fixation rates were low (average 0.63 ± 0.07 nmol N L−1 d−1), but consistently detected across all depths and stations, representing ~ 6–88 % of photic N2 fixation. N2 fixation rates were not significantly correlated to DOM compounds. The analysis of nifH gene amplicons revealed a wide diversity of non-cyanobacterial diazotrophs, majorly matching clusters 1 and 3. Interestingly, a distinct phylotype from the major nifH subcluster 1G dominated at 650 dbar, coinciding with the oxygenated Sub-Antarctic Mode Water (SAMW). This consistent pattern suggests that the distribution of aphotic diazotroph communities is to some extent controlled by water mass structure. While the data available is still too scarce to elucidate the distribution and controls of mesopelagic non-cyanobacterial diazotrophs in the WTSP, their prevalence in the mesopelagic layer and the consistent detection of active N2 fixation activity at all depths sampled during our study suggest that aphotic N2 fixation may contribute significantly to fixed nitrogen inputs in this area.