The effect of inorganic nutrients on planktonic assemblages has been traditionally relied on concentrations rather than estimates of nutrient supply. We combined a novel dataset of hydrographic properties, turbulent mixing, nutrient concentration and picoplankton community composition with the aim of: i) quantifying the role of temperature, light and nitrate fluxes as factors controlling the distribution of autotrophic and heterotrophic picoplankton subgroups, as determined by flow cytometry; and ii) describing the ecological niches of the various components of the picoplankton community. Data were collected in 97 stations in the Atlantic Ocean, including tropical and subtropical open ocean waters, the northwestern Mediterranean Sea, and the Galician coastal upwelling system of the Northwest Iberian Peninsula. A Generalized Additive Model (GAM) approach was used to predict depth-integrated biomass of each picoplankton subgroup based on three niche predictors: sea surface temperature, averaged daily surface irradiance, and the transport of nitrate into the euphotic zone, through both diffusion and advection. In addition, niche overlap between different picoplankton subgroups was computed using non-parametric kernel density functions. Temperature and nitrate supply were more relevant than light in predicting the biomass of most picoplankton subgroups, except for Prochlorococcus and low nucleic acid (LNA) prokaryotes, for which irradiance also played a significant role. Nitrate supply was the only factor that allowed the distinction between the ecological niches of all autotrophic and heterotrophic picoplankton subgroups. Prochlorococcus and LNA prokaryotes were more abundant in warmer waters (> 20 ºC) where the nitrate fluxes were low, whereas Synechococcus and high nucleic acid (HNA) prokaryotes prevailed mainly in cooler environments characterized by intermediate or high levels of nitrate supply. Finally, the niche of picoeukaryotes was defined by lower temperatures and high nitrate supply. We estimated that with the predicted decrease in nitrate supply as the result of global change, a 9 % increase in the ratio cyanobacteria to picoeukaryotes would occur in a future ocean, something that would have important implications, given the contribution of smallest cells to the biological carbon pump. These results support the key role of nitrate supply, as it not only promotes the growth of large phytoplankton, but it also controls the structure of marine picoplankton communities.