The aim of the study was to determine mycoplankton species diversity in relation to the physico-chemical parameters of lake waters. The research was carried out in the summer months in 15 mesotrophic lakes and showed a high ecological significance index for Rhodotorula glutinis, Epicoccum nigrum, Fusarium sporotrichioides, and Trichophyton violaceum. Mycoplankton abundance and species diversity decreased with the depth of water, which coincided with a decrease in oxygen content and organic matter concentration. A high concentration of nitrogen compounds (total nitrogen—TN and dissolved nitrogen—DN) limited the development of mycobiota in the hypolimnion. In the metalimnion, the intensive development of organisms, especially bacteria, limited mycoplankton abundance despite perfect physical and chemical conditions for its development. Finally, mycoplankton functioned the best in slightly alkaline waters.