Mesoscale oceanic eddies contribute to the redistribution of resources needed for plankton to thrive. However, due to their fluid-trapping capacity, they can also isolate plankton communities, subjecting them to rapidly changing environmental conditions. Diazotrophs, which fix dinitrogen (N2), are key members of the plankton community, providing reactive nitrogen, particularly in large nutrient-depleted regions such as subtropical gyres. However, there is still limited knowledge about how mesoscale structures characterized by specific local environmental conditions can affect the distribution and metabolic response of diazotrophs when compared with the large-scale dynamics of an oceanic region. Here we investigated genetic diazotroph diversity and N2 fixation rates in a transect across the Gulf Stream and two associated eddies, a region with intense mesoscale activity known for its important role in nutrient transport into the North Atlantic Gyre. We show that eddy edges are hotspots for diazotroph activity with potential community connectivity between eddies. Using a long-term mesoscale eddy database, we quantified N2 fixation rates as up to 17 times higher within eddies than in ambient waters, overall providing ~21 µmol N m−2 yr−1 to the region. Our results indicate that mesoscale eddies are hotspots of reactive nitrogen production within the broader marine nitrogen cycle.