This study presents an original rDNA PCR and microscopic survey of pelagic freshwater fungal communities, and was designed to unveil the diversity of true Fungi (i.e. the kingdom Eumycota) in three contrasting lake ecosystems (Lakes Pavin, Aydat and Vassivière) located in the French Massif Central. Three clone libraries were constructed from samples collected in the euphotic layers of the lakes during spring 2007. Phylogenetic analysis of the combined data from the three lakes clustered our sequences into thee divisions: Chytridiomycota (50% of total sequences), Ascomycota (40%) and Basidiomycota (10% in Pavin and Aydat only). Several sequences were assigned to a novel Chytridiomycota clade first recovered in Lake Pavin in 2005. Most of the sequences retrieved in the investigated lakes were affiliated with known fungal species, most of which were apparently well adapted to thrive in the pelagic realm. Their main functions (i.e. parasitism and saprophytism), putatively inferred from the closest relatives of the retrieved molecular sequences, were confirmed by microscopic approaches and by enrichment experiments with pollen grains. The occurrence of three fungal forms (zoosporic, yeast and mycelial) was associated with different trophic modes, establishing fungi as strong potential competitors for various niches in pelagic ecosystems, primarily in relation to the processing of particulate organic matter and the production of propagule food sources for grazers. For the first time, this study provides insight into the diversity and the associated functions of all members of the Kingdom Eumycota investigated in the whole plankton fraction of aquatic ecosystems.