[1] Biological N₂ fixation rates were quantified in the Eastern Tropical South Pacific (ETSP) during both El Niño (February 2010) and La Niña (March–April 2011) conditions, and from Low‐Nutrient, Low‐Chlorophyll (20°S) to High‐Nutrient, Low‐Chlorophyll (HNLC) (10°S) conditions. N₂ fixation was detected at all stations with rates ranging from 0.01 to 0.88 nmol N L⁻¹ d⁻¹, with higher rates measured during El Niño conditions compared to La Niña. High N₂ fixations rates were reported at northern stations (HNLC conditions) at the oxycline and in the oxygen minimum zone (OMZ), despite nitrate concentrations up to 30 µmol L⁻¹, indicating that inputs of new N can occur in parallel with N loss processes in OMZs. Water‐column integrated N₂ fixation rates ranged from 4 to 53 µmol N m⁻² d⁻¹ at northern stations, and from 0 to 148 µmol m⁻² d⁻¹ at southern stations, which are of the same order of magnitude as N₂ fixation rates measured in the oligotrophic ocean. N₂ fixation rates responded significantly to Fe and organic carbon additions in the surface HNLC waters, and surprisingly by concomitant Fe and N additions in surface waters at the edge of the subtropical gyre. Recent studies have highlighted the predominance of heterotrophic diazotrophs in this area, and we hypothesize that N₂ fixation could be directly limited by inorganic nutrient availability, or indirectly through the stimulation of primary production and the subsequent excretion of dissolved organic matter and/or the formation of micro‐environments favorable for heterotrophic N₂ fixation.