Lymantria dispar (gypsy moth), one of the most destructive forest pests, causes periodic outbreaks culminating with an epizootic of the baculovirus L. dispar multiple nucleopolyhedrovirus (LdMNPV). However, the source and mode of LdMNPV infection remain unclear. To examine whether LdMNPV in the soil acts as an inoculum source for an epizootic, we performed DNA analyses and bioassays using samples collected in Yamanashi, Japan. In the study area, L. dispar outbreak began in 2019 and an LdMNPV epizootic occurred in 2020. Using purified occlusion bodies (OBs), the LC50 value of the droplet-feeding bioassay was 1.9 × 105 OBs/mL and that of the soil-feeding bioassay was 6.4 × 104 OBs/mL. PCR assay could detect purified OBs dose-dependently but not soil-mixed OBs. Soil-feeding bioassays using field-collected soils and PCR assay revealed that LdMNPV OBs maintained their biological potency and were estimated to be approximately 104 OBs/g soil. Larvae fed soil from after the epizootic showed a significant lower survival rate than ones fed soil from before the epizootic. DNA sequencing showed increased LdMNPV genetic diversity in the same locality and a single host during the epizootic, suggesting the migration of LdMNPV-infected larvae into the study site. Furthermore, a few identical LdMNPV haplotype were dominant during the epizootic, and the pathogenicity such as mortality rate and the OBs yields did not significantly differ between the LdMNPV isolates. These results suggest that LdMNPV in the soil is an important source of inoculum infecting L. dispar.