Cyanobacterial harmful algal blooms (or CyanoHABs) represent one of the most conspicuous waterborne microbial hazards. The characterization of the bloom communities remains problematic because the cyanobacterial taxonomy of certain genera has not yet been resolved. In this study, 29 planktic and benthic cyanobacterial strains were isolated from freshwaters located in Greece. The strains were assigned to the genera Chroococcus, Microcystis, Synechococcus, Jaaginema, Limnothrix, Pseudanabaena, Anabaena, and Calothrix and screened for the production of the cyanotoxins microcystins (MCs), cylindrospermopsins (CYNs), and saxitoxins (STXs) using molecular (PCR amplification of seven genes implicated in cyanotoxin biosynthesis) and immunological (ELISA) methods. This study presents, for the first time, a cyanobacteria culture collection from Greece, thus providing missing study material for the understanding of bloom formation and cyanotoxin production in the Mediterranean and for the polyphasic characterization of important components of the phytoplankton. The combined use of molecular and immunochemical methods allowed the identification of MC producing strains, but further data are needed for CYNand STX-producing cyanobacteria. The high percentage of MC-producing Microcystis strains in the urban Lakes Kastoria and Pamvotis, frequently used for agriculture irrigation, fishing and recreation, highlights the potential risk for human health.