Freshwater fungi are a poorly studied paraphyletic group that include a high diversity of phyla. Most studies of aquatic fungal diversity have focussed on single habitats, thus the linkage between habitat heterogeneity and fungal diversity remains largely unexplored. We took 216 samples from 54 locations representing eight different habitats in meso-oligotrophic, temperate Lake Stechlin in northern Germany, including the pelagic and littoral water column, sediments, and biotic substrates. We pyrosequenced a universal eukaryotic marker within the ribosomal large subunit (LSU) in order to compare fungal diversity, community structure, and species turnover among habitats. Our analysis recovered 1024 fungal OTUs (97% criterion). Diversity was highest in the sediment, biofilms, and benthic samples (293-428 OTUs), intermediate in water and reed samples (36-64 OTUs), and lowest in plankton (8 OTUs) samples. NMDS clustering clearly grouped the eight studied habitats into six clusters, indicating that total diversity was strongly influenced by turnover among habitats. Fungal communities exhibited pronounced changes at the levels of phylum and order along a gradient from littoral to pelagic habitats. The large majority of OTUs could not be classified below the order level due to the lack of aquatic fungal entries in taxonomic databases. Our study provides a first estimate of lake-wide fungal diversity and highlights the important contribution of habitat-specificity to total fungal diversity. This remarkable diversity is probably an underestimate, because most lakes undergo seasonal changes and previous studies have uncovered differences in fungal communities among lakes.