Nitrogen fixation is no longer considered to be a minor factor of the nitrogen cycle in oceanic ecosystems. Recent geochemical and biological efforts have led to a significant increase in the estimated input of nitrogen to marine ecosystems by biological fixation, while molecular studies have increased our knowledge of the number and diversity of nitrogen fixers known to be active in the ocean. Although Trichodesmium spp. have long been viewed as the primary marine nitrogen fixers, recent efforts have shown that various members of the picoplankton community are also actively involved in nitrogen fixation. The relative abundance of different nitrogen fixers is an important ecosystem parameter since nitrogen fixers may differ significantly in their physiology, life history and ecology. Here we combine rate measurements and stable isotope natural abundance measurements to constrain the impact of N2 fixation in the waters north of Australia. Samples were collected in the Coral, Arafura, and East Timor Seas, thus spanning three distinct hydrographic regions. Our data show that Trichodesmium has a significant influence on the stable nitrogen isotope ratios of particulate and zooplankton biomass and suggest that Trichodesmium is a significant source of nitrogen for the pelagic ecosystem. Based on stable carbon isotope ratios, it is also likely that the pathways are indirect and nitrogen fixed by Trichodesmium enters the higher trophic levels via decomposition as dissolved organic and inorganic nitrogen. Picocyanobacteria showed high diazotrophic activity at some stations, but unlike Trichodesmium, their N2 fixation rate was not reflected in the stable N isotope ratios of particulate and zooplankton biomass. Our results suggest an important N contribution to biomass by diazotrophs in the Coral Sea, Arafura Sea and East Timor Sea.