The role of nitrogen cycling in submarine hydrothermal systems is far less studied than that of other biologically reactive elements such as sulfur and iron. In order to address this knowledge gap, we investigated nitrogen redox processes at Loihi Seamount, Hawaii, using a combination of biogeochemical and isotopic measurements, bioenergetic calculations and analysis of the prokaryotic community composition in venting fluids sampled during four cruises in 2006, 2008, 2009 and 2013. Concentrations of NH4+ were positively correlated to dissolved Si and negatively correlated to NO3–+NO2–, while NO2– was not correlated to NO3–+NO2–, dissolved Si or NH4+. This is indicative of hydrothermal input of NH4+ and biological mediation influencing NO2–concentrations. The stable isotope ratios of NO3– (δ15N and δ18O) was elevated with respect to background seawater, with δ18O values exhibiting larger changes than corresponding δ15N values, reflecting the occurrence of both production and reduction of NO3– by an active microbial community. δ15N-NH4+ values ranged from 0‰ to +16.7‰, suggesting fractionation during consumption and potentially N-fixation as well. Bioenergetic calculations reveal that several catabolic strategies involving the reduction of NO3– and NO2– coupled to sulfide and iron oxidation could provide energy to microbes in Loihi fluids, while 16S rRNA gene sequencing of Archaea and Bacteria in the fluids reveals groups known to participate in denitrification and N-fixation. Taken together, our data support the hypothesis that microbes are mediating N-based redox processes in venting hydrothermal fluids at Loihi Seamount.