Pico- and nanoplanktonic protists (eukaryotic microorganisms with cell size of <3 μm and 3–20 μm, respectively) are the key component of plankton communities. However, their diversity and distribution patterns along environmental factors are still poorly recognized, largely due to their very large phylogenetic diversity that has been determined only via the application of molecular methods over the past two decades. Here, we compared diversity and composition of active communities of pico- and nanoplantonic protists from three zones of the Vistula River estuary (Gulf of Gdańsk): freshwater, mixing (salinity 3.5) and brackish (salinity 7), in four seasons, by pyrosequencing the V3-V4 fragment of 18S rRNA taxonomy marker gene libraries. Alpha diversity was the highest at the brackish site, but the OTU (Operational Taxonomic Units) richness was characteristic for specific protist groups at each site. The active protistan communities in the freshwater and mixing zones (salinity 0–3.5) were similar (sharing >72% of phylotypes) and included centric diatoms (Stephanodiscus minutulus), synurophytes from clades C, E and F, and cryptophytes. However, at salinity of 7 at the brackish site the communities were significantly different from those in freshwater/mixing zone, and showed higher contributions of Dinophyceae, Mamiellophyceae, Telonemia, and picobiliphytes. The high similarity between the freshwater and mixing site, as well as high dissimilarity of the brackish site was observed in all months, despite seasonal shifts in pico- and nanoplantonic protistan communities. Seventy five percent of the observed variability in the communities was explained by combinations of temperature, salinity, nutrients and geographical distance, indicating interplay between species sorting and mass effects in shaping the active protistan communities in the Vistula River estuary. Groups that were more active in freshwaters and the mixing zone seemed to be more affected by mass effects of mixing water masses, while those from the brackish site by environmental species sorting. Finally, we report, for the first time, presence of Radiolaria (Acantharea) from the Baltic Sea.