Oomycetes play a key role in decomposing and recycling organic materials in freshwater ecosystems. However, their diversity and ecological study gained less attention compared with other organisms. Based on extensive, long-term surveys for freshwater oomycetes, the present study undertakes a comprehensive examination of oomycete diversity and ecology in the Korean freshwater environments. We successfully identified a total of 78 taxa of oomycetes (568 isolates), which are classified into the orders Peronosporales (4%), Pythiales (77%), and Saprolegniales (19%), based on their cultural, morphological, and molecular characteristics. These taxa consist of 57 previously described species, including 22 species new to Korea, and 21 previously unknown lineages. Eight of these taxa were described as new species under the Pythiales, with detailed taxonomic descriptions in this study. Our findings revealed that the distributions of freshwater oomycetes varied depending on substrates and that oomycetes were primarily found in plant-derived sources, such as decaying leaves, stems, and twigs. Across all seasons and freshwater ecosystem types, Pythiales were more frequently isolated than Peronosporales and Saprolegniales. However, isolation frequencies among taxa varied across seasons and habitat types. This study provides extensive strain resources and establishes a fundamental reference for future taxonomic and ecological research on freshwater oomycetes, expanding knowledge of their diversity and functional roles in freshwater ecosystems.