ABSTRACT: The contribution of planktonic diazotrophs to the overall N budget is a key unknown in the eastern Indian Ocean. Here we investigated the relationships between dissolved inorganic nutrients, phytoplankton pigment composition, microbial community structure, nitrogen fixation rates and the δ15N of fractionated zooplankton samples along the shelf break of Western Australia (32° to 12° S) in September 2012. Bulk nitrogen fixation rates declined from 4.8 nmol l−1 h−1 in the colder and more saline sub-tropical waters at higher latitudes to 1.5 nmol l−1 h−1 in the warmer and fresher Timor Sea at lower latitudes. A regional bloom of Trichodesmium was identified between 13° and 9° S in the Timor Sea. Trichodesmium-specific N2 fixation rates were 0.05 ± 0.01 nmol colony−1 h−1. Highest dissolved inorganic nitrogen (DIN) concentrations occurred at the highest NH4+:NO3− ratios, thereby deviating from the paradigm that greater DIN concentrations come primarily from increased NO3− through advection, mixing or upwelling. Both the microplankton and nanoflagellate fraction declined significantly in warmer waters, with higher DIN concentrations but decreasing % NO3−. A clear increase in the prokaryotic diagnostic pigment zeaxanthin was seen with increasing temperatures from higher to lower latitudes. The microbial community, measured using automated ribosomal intergenic spacer analysis (ARISA), clustered strongly according to the water mass biogeochemistry including temperature, salinity, DIN and phosphate concentrations (p < 0.001). Isotope analysis suggested that injections of low δ15N from N2 fixation lowered the zooplankton δ15N signature of animals up to ~500 μm in size and that nearly 47% of the fixed nitrogen was used by zooplankton (≤500 μm fraction) in the Timor Sea.