Bacillus subtilis bacteria have been described as an important starter culture in fermented soybean foods. An earlier study isolated a lytic BasuTN3 bacteriophage effective against the B. subtilis strain TN3 isolated from Thua Nao, a Thai fermented soybean product. The bacteriophage infection is a persistent threat that can cause delayed or failed fermentations. In the present study, four bacterial strains (TN3, DM1-2, and FM2-3 isolated from Thua Nao, and ASA from Natto)—which could be considered as potential inocula for Thua Nao fermentation based on their relative index values of protease activity—are vulnerable to the BasuTN3 phage, as demonstrated by the double layer assay. These four strains were then phenotypically and genotypically characterized; these included morphology, biochemical assays (e.g., IMViC test), fermentation of carbohydrates using API 50 CHB, and 16S rRNA gene sequence. These four bacterial isolates were rod-shaped, endospore-forming, Gram-positive bacteria. Based on the biochemical assays, all isolates exhibited similar profiles (not identical). Further molecular analysis using the 16S rRNA gene sequence revealed that they belonged to a distinct group of B. subtilis. It should be noted, however, that these four strains were distinct based on their overall characteristics, which could be considered as ‘strain-dependent’. The results of the present work are expected to be useful for comparative purposes of the key characteristics (both phenotypic and genotypic) among the closely related Bacillus strains. These data would be useful for future screening and isolation of the bacterial inocula.