The main objective of this work was to determine the abundance and species diversity of fungi in the waters of selected rivers of Central Europe, NE Poland (Augustów Lakeland), differing in size, physical and chemical properties, and streamflow rate. The minimum abundance of fungi in the analysed rivers was recorded for a river with low concentration of organic matter (8200 CFU/mL, Czarna Hańcza River), and maximum for a strongly anthropogenically polluted river (24,800 CFU/mL, Kamienny Bród River). A total of 49 fungal species were identified based on PCR ITS-RFLP and DNA sequencing methods. However, RFLP-PCR method has proved to be sufficient to determine the species of 34 fungi. The highest taxonomic diversity was determined for the waters abundant in organic matter (Piecówka and Rospuda Rivers), and the lowest for rivers poor in organic matter (Netta and Czarna Hańcza Rivers). From the 49 identified species, 47% were aquatic hyphomycetes, and 18% were potentially pathogenic fungi mainly occurring in the waters of polluted rivers with increased organic matter concentrations. Moreover, a higher number of fungal taxa were recorded in fluvial waters distinguished by higher streamflow rate, and therefore, stronger water turbulence. The study results suggest that the most important factors influencing the structure of mycoplankton in rivers include pH of water, content of organic matter, degree of anthropogenic pollution, and streamflow rate.