Esteya vermicola, as the first recorded endoparasitic fungus of pinewood nematodes, exhibits great potential as a biological agent against nematodes. However, only two strains of this species have been described so far. In this study, we identified a novel endoparasitic fungal strain, CNU 120806, isolated from infected nematodes in forest soil samples during a survey of nematophagous fungi in Korea. This strain showed similar morphological characteristics and infection mode with the two previously described strains of E. vermicola. All strains are characterized by the ability to produce two types of conidiogenous cells and conidia, and to parasitize nematodes with lunate adhesive conidia. Moreover, the CNU 120806 strain showed 100% identity with E. vermicola CBS 115803 when their partial sequences of 28S rRNA gene were compared. Molecular phylogenetic analysis further identified CNU 120806 as a strain of E. vermicola, by clustering CNU 120806 and E. vermicola CBS 115803 into a single subclade. Culture medium influenced the proportion of dimorphic CNU 120806 conidia, and further changed the adhesive and mortality rates of nematodes. The CNU 120806 strain exhibits high infection activity against nematodes on nutrient-rich PDA medium. Almost all tested nematodes were killed within 8~10 days after inoculation. This study provides justification for further research of E. vermicola, and the application and formulation of this fungus as a bio-control agent against nematodes.