Dinitrogen (N2) fixers (diazotrophs) fuel primary productivity by providing reactive nitrogen into the ocean ecosystem and promoting CO2 sequestration. N2 fixation has been extensively studied in the low latitudes of the Atlantic and Pacific Oceans. By comparison, the Indian Ocean remains the least explored and most enigmatic ocean basin. This is particularly the case for the Southern Indian Ocean (SIO). Here we explore N2 fixation activity and diazotroph community composition, diversity, and abundance from 20 to 60ºS in the SIO. While this region plays a key biogeochemical role serving as a link between the Atlantic and South Pacific Ocean waters, its N2 fixation potential remains unknown. Our results provide new insights into diazotrophy in a poorly studied region and expand the range of biomes where diazotrophy may be observed.