The abundances of six N2-fixing cyanobacterial phylotypes were profiled at 22 stations across the tropical Atlantic Ocean during June 2006, and used to model the contribution of the diazotrophs to N2 fixation. Diazotroph abundances were measured by targeting the nifH gene of Trichodesmium, unicellular groups A, B, C (UCYN-A, UCYN-B and UCYN-C), and diatom-cyanobiont symbioses Hemiaulus–Richelia, Rhizosolenia–Richelia and Chaetoceros–Calothrix. West to east gradients in temperature, salinity and nutrients [NO3- + NO2-, PO43−, Si(OH)4] showed the influence of the Amazon River plume and its effect on the distributions of the diazotrophs. Trichodesmium accounted for more than 93% of all nifH genes detected, dominated the warmer waters of the western Atlantic, and was the only diazotroph detected at the equatorial upwelling station. UCYN-A was the next most abundant (> 5% of all nifH genes) and dominated the cooler waters of the eastern Atlantic near the Cape Verde Islands. UCYN-C was found at a single depth (200 m) of high salinity and low temperature and nutrients, whereas UCYN-B cells were widespread but in very low abundance (6.1 × 101 ± 4.6 × 102 gene copies l−1). The diatom-cyanobionts were observed primarily in the western Atlantic within or near the high Si(OH)4 input of the Amazon River plume. Overall, highest diazotroph abundances were observed at the surface and declined with depth, except for some subsurface peaks in Trichodesmium, UCYN-B and UCYN-A. Modelled contributions of Trichodesmium, UCYN-B and UCYN-A to total N2 fixation suggested that Trichodesmium had the largest input, except for the potential of UCYN-A at the Cape Verde Islands.