The influence of irradiance, temperature, and NO3 concentration over the biomass, growth rate, and primary production rate of picophytoplankton was assessed in an inshore to offshore gradient in the temperate waters of the southern Bay of Biscay (NE Atlantic). Moreover, we analysed the effect of these variables on the seasonal succession of broad picophytoplankton community structure (i.e. Synechococcus, Prochlorococcus, small, and large picoeukaryotes). Picophytoplankton showed higher biomasses, primary production rates, and growth rates inshore, which were associated with higher irradiances and NO3 concentrations in these mixed layers as compared to offshore locations. Seasonally, picophytoplankton presented high biomasses in summer, growth rates in spring, and primary productions in autumn. Most picophytoplankton biomass was represented by picoeukaryotes inshore, 70.8%, whilst cyanobacteria were more important offshore, 42.3%. Seasonally, picoeukaryotes were the higher contributors to picophytoplankton biomass in spring and winter, nearly 75%. Moreover, small picoeukaryotes represented nearly 50% of the picophytoplankton biomass in winter, and the greater presence of large picoeukaryotes was found in spring, nearly 35%. Conversely, cyanobacteria peaked in summer, 55.4%, with Synechococcus amounting to 52.4% of picophytoplankton biomass. Maximum contribution of Prochlorococcus was found in autumn, 10.4%. A multiple regression model was used to analyze the association between the biomass of different groups of picophytoplankton in the mixed layer and its irradiance, temperature, and the interaction between irradiance and temperature. This shed light on the ecological conditions affecting the community structure of a planktonic size-class traditionally regarded as homogeneous. Finally, an intaglio representation showed the seasonal succession of picophytoplankton.