We measured rates of N- and C-fixation with a direct tracer method in regions of the western tropical North Atlantic influenced by the Amazon River plume during the high flow period of 2010 (May–June 2010). We found distinct regional variations in N-fixation activity, with the lowest rates in the plume proper and the highest rates in the plume margins and in offshore waters. A comparison of our N- and C-fixation measurements showed that the relative contribution of N-fixation to total primary production increased from the plume core toward oceanic waters, and that most of the C-fixation in this system was supported by sources of nitrogen other than those derived from biological N-fixation, or diazotrophy. We complemented these rate experiments with measurements of the δ15N of suspended particles (δ15PN), which documented the important and often dominant role of diazotrophs in supplying nitrogen to particulate organic matter in the water column. These coupled measurements revealed that small phytoplankton contributed more new nitrogen to the particulate nitrogen pool than larger phytoplankton. We used a habitat classification method to assess the fac- tors that control diazotrophic activity and contribution to the suspended particle pool, both of which increased from the plume toward oceanic waters. Our findings provide an important constraint on the role of the Amazon plume in creating distinct niches and roles for diazotrophs in the nutrient and carbon budgets of the western tropical North Atlantic.