Understanding the distribution of the different picoplankton groups represents a central tenet of marine microbial ecology. Centering our study on the three major groups constituting the bulk picoplankton community (size 0.2-3 mm), we sought to analyze the distribution of autotrophic bacteria (Synechococcus and Prochlorococcus), photosynthetic Picoeukaryotes pPeuk, and heterotrophic bacteria. For that objective, two different strategies were used, the first one was based on flow cytometry for determining ataxonomic patterns in picoplankton distribution, and the second a comparative analysis approach for identifying broad patterns in bacterial phylogenetic community structure. Given that conversion factors (CFs) were necessary to translate group cell abundance into carbon biomass, but that large discrepancies for CF values of pPeuk had been reported in the literature, we first (re-) evaluated the CF for small phototrophic picoeukaryotes (<5 mm). On one hand, as the set of cultures of Peuk used for that purpose were maintained in non-axenic conditions, we compared two different methods for correcting errors in biomass estimation due to presence of bacteria. Secondly, a relatively higher CF value for pPeuk than those previously reported to date was found, with implications on the role generally attributed to pPeuk in the carbon cycling and other ecosystem processes. Applying this CF, we could identify patterns of variability in picoplankton group distribution at different spatio-temporal scales during winter in a NW Mediterranean coastal station and during a cruise performed in summer from coast to offshore off the Catalan coast. By focusing on the variability at the short time scale, our work showed not only evidences of coupling between picophytoplankton variability and the single-cell bacterial activities but also highlighted how a relatively small variation in meteorology changed considerably the structure of the microbial community. Different trends of variability were observed between the different picoplankton groups, pPeuk cell numbers exhibiting the highest spatio-temporal variability, and bacterial abundance the lowest. Opposite patterns between picoplankton community structure and chlorophyll a levels were observed not only spatially, but also at both the short-term and large temporal scale, suggesting that picoplankton group distribution are useful indicators of the ecosystem state. Finally, we assessed the biogeography of the bacterial phylogenetic groups along a continuum of environmental parameters such as chlorophyll a, temperature and salinity, and identified different patterns in bacterial community structure as related to phytoplankton biomass among coastal and open ocean ecosystems, suggesting unequal metabolic aptitudes of the different bacterial groups for utilizing algal-derived DOC