The establishment of a laboratory assay and a greenhouse assay was conducted for evaluating the pathogenicity of Macrophomina phaseolina causing soybean charcoal rot established. In the laboratory assay, microsclerotia and hyphae were used as inoculum. In the laboratory assays using microsclerotia as an inoculum, disease incidences of M. phaseolina NSW17-108 and HSM17-034 were higher at 35°C than 25°C. Of the two isolates NSW17-108 and HSM17-034, the disease incidence of HSM17-034 isolated from diseased sesame is higher than that of NSW17-108 isolated from diseased soybean. When the mycelia of M. phaseolina were used as an inoculum, the disease incidence of NSW17-108 and HSM17-034 at 35°C exceeded 80% even after only 5 days of inoculation. Even at 25°C, furthermore, that of HSM17-034 exceeded 80% 5 days later. In the pathogenicity assays at a greenhouse, toothpicks where microsclerotia were produced or microsclerotia harvested from potato dextrose agar medium were used as an inoculum. In all greenhouse assays, M. phaseolina NSW17-108 and HSM17-034 showed 40–60% of disease incidences 35–65 days after inoculation with the pathogen, depending on the inoculation method. Between the two isolates, the pathogenicity of HSM17-034 was stronger than that of NSW17-108, and this result was consistent with laboratory assay results. Since the laboratory and greenhouse test methods tested in this study have different advantages and disadvantages depending on each test method, it is thought that the test method that can meet the purpose of the study should be selected and used.