Ascomycota with 93,000 known species, is the largest phylum of the Fungi and one of the most ubiquitous and diverse phyla of eukaryotes. Many species in the phylum are great importance for their application in biotechnology and bioindustry. However, a number of species in Korea is still poorly known. In a survey of fungal diversity in freshwater niche in Korea, three strains were isolated from freshwater samples collected at Deudeul river located in Naju, Jeonnam Province, and at Cheongyang, Chungnam Province, Korea. Based on the morphological characteristics and combined analysis of the internal transcribed spacer (ITS) regions, RNA polymerase II subunit (RPB2) and β-tubulin (TUB2) gene, three strains were identified as Clonostachys eriocamporesii, Penicillium anthracinoglaciei and Trichoderma macrofasciculatum. To our knowledge, these species have never been previously described in Korea.