This study aimed to isolate and characterize fungi from freshwater environments in South Korea and evaluate their extracellular enzyme activities. Fungal strains were collected from various freshwater sources and identified using phylogenetic analysis. The isolated fungi included known aquatic hyphomycetes and previously unreported species. Extracellular enzyme, including those of protease, amylase, lipase, cellulase, laccase, and chitinase, activities were evaluated. Among the isolated strains, several showed high enzyme activity, suggesting their potential role in organic matter decomposition in freshwater ecosystems. This research expands our knowledge of the diversity and enzyme activities of the fungi in freshwater environments, contributing to our understanding of their ecological roles.