The contribution of picocyanobacteria to summer phytoplankton blooms, accompanied by an ecological crisis, is a new phenomenon in Europe. This issue requires careful investigation. We studied allelopathic activity of freshwater picocyanobacterium Synechococcus sp. on phytoplankton assemblages from three freshwater lakes. In this study, the allelopathic activity of the Synechococcus sp. on the total abundance, biomass, as well as structure of the phytoplankton assemblages were investigated. Our results indicated that addition of exudates obtained from Synechococcus sp. affected the number of cells and biomass of the phytoplankton communities; the degree of inhibition or stimulation was different for each species, causing a change in the phytoplankton abundance and dominance during the experiment. We observed that some group of organisms (especially cyanobacteria from the genus Aphanothece, Limnothrix, Microcystis, and Synechococcus) showed tolerance for allelopathic compounds produced and released by Synechococcus sp. It is also worth noting that in some samples, Bacillariophyceae (e.g., Amphora pediculus, Navicula pygmaea, and Nitzschia paleacea) were completely eliminated in the experimental treatments, while present in the controls. This work demonstrated that the allelopathic activity exhibited by the Synechococcus sp. is probably one of the major competitive strategies affecting some of the coexisting phytoplankton species in freshwater ecosystems. To our best knowledge this is the first report of the allelopathic activity of Synechococcus sp. in the freshwater reservoirs, and one of the few published works showing allelopathic properties of freshwater picocyanobacteria on coexisting phytoplankton species.