Data concerning picoplanktonic community composition and abundance in the Central Adriatic Sea are presented in an effort to improve the knowledge of bacterioplankton and autotrophic picoplankton and their seasonal changes. Flow cytometry analyses revealed the presence of two distinct bacteria populations: HNA and LNA cells. HNA cells showed an explicit correlation with viable and actively respiring cells. The study of viability and activity may increase our knowledge of the part that contributes really to the remineralization and bacterial biomass production. Authotrophic picoplankton abundance, especially picocyanobacteria, was strongly influenced by seasonality, indicating that light availability and water temperature are very important regulating factors. In terms of total carbon biomass, the main contribution came from heterotrophic bacteria with a lower contribution from autotrophic picoplankton. CARD-FISH evidenced, within the Eubacteria domain, the dominance of members of the phyla Alphaproteobacteria, with a strong contribution from SAR11clade, followed by Cytophaga-Flavobacterium and Gammaproteobacteria. The bacterial groups detected contributed differently depending when the sample was taken, suggesting possible seasonal patterns. This study documents for the first time picoplankton community composition in the Central Adriatic Sea using two different approaches, FCM and CARD-FISH, and could provide preliminary data for future studies.