Zoosporic fungi play an important role in aquatic environments, but their diversity, especially that of parasitic fungi of phytoplankton, has still not been fully revealed. We conducted monthly analyses of the community structure of zoosporic fungi at a pelagic site in Lake Biwa, Japan, from May to December 2016. Metabarcoding analysis, targeted to a large subunit region of ribosomal DNA in the nano-size fraction of particles (2-20 µm), was carried out on the samples. We also counted large phytoplankton and chytrid sporangia attached to the hosts. We detected 3 zoosporic fungal phyla (Blastocladiomycota, Chytridiomycota and Cryptomycota) within 107 operational taxonomic units (OTUs), in which Chytridiomycota was the most diverse and abundant phylum. Few fungal OTUs overlapped between months, and specific communities were detected in each month. These results showed that diverse zoosporic fungi with high temporal variability inhabited the lake. Five large phytoplankton species were found to be infected by chytrids: Staurastrum dorsidentiferum, S. rotula, Closterium aciculare, Asterionella formosa and Aulacoseira granulata. Some chytrids were detected by metabarcoding analysis: Zygophlyctis asterionellae infecting A. formosa, Staurastromyces oculus infecting S. dorsidentiferum and Pendulichytrium sphaericum infecting A. granulata. One OTU detected in association with infected C. aciculare by microscopic counting might have been an obligate parasitic chytrid of C. aciculare. The results indicated that a combination of metabarcoding and microscopic analysis revealed more information on zoosporic fungi, including those that are parasitic.