Trichoderma atroviride is a common fungus found in various ecosystems that shows mycoparasitic ability on other fungi. A novel dsRNA virus was isolated from T. atroviride NFCF377 strain and its molecular features were analyzed. The viral genome consists of a single segmented double-stranded RNA and is 9,584 bp in length, with two discontinuous open reading frames (ORF1 and ORF2). A mycoviral structural protein and an RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively, between which is found a canonical shifty heptameric signal motif (AAAAAAC) followed by an RNA pseudoknot. Analysis of sequence similarity and phylogeny showed that it is closely related to members of the proposed family “Fusagraviridae”, with a highest similarity to the Trichoderma atroviride mycovirus 1 (TaMV1). Although the sequence similarity of deduced amino acid to TaMV1 was evident, sequence deviations were distinctive at untranslated regions (UTRs) due to the extended size. Thus, we inferred this dsRNA to be a different strain of Trichoderma atroviride mycovirus 1 (TaMV1-NFCF377). Electron microscopy image exhibited an icosahedral viral particle of 40 nm diameter. Virus-cured isogenic isolates were generated and no differences in growth rate, colony morphology, or conidia production were observed between virus-infected and virus-cured strains. However, culture filtrates of TaMV1- NFCF377-infected strain showed enhanced antifungal activity against the plant pathogen Rhizoctonia solani but not to edible mushroom Pleurotus ostreatus. These results suggested that TaMV1-NFCF377 affected the metabolism of the fungal host to potentiate antifungal compounds against a plant pathogen, but this enhanced antifungal activity appeared to be species-specific.