Although many Bacillus species are known to be good antibiotic producers capable of acting as biocontrol agents, the underlying antimicrobial mechanisms are often poorly understood. In this study, 21 Bacillus strains, demonstrating over 50% mycelial inhibition against Sclerotinia sclerotiorum as well as significant control in plant assays, were examined for the presence of antibiotic biosynthetic genes. Primers specific for bacillomycin D, iturin A, surfactin, mycosubtilin, fengycin, and zwittermicin A were used to amplify biosynthetic genes from these bacteria using PCR. The majority of strains harbored surfactin (21/21) and iturin A (20/21) biosynthetic genes. Three strains (Bacillus subtilis 3057, Bacillus amyloliquefaciens BS6, and Bacillus mycoides 4079) were positive for bacillomycin D, whereas 4 strains (B. subtilis H-08-02, B. subtilis 3057, B. amyloliquefaciens BS6, and B. mycoides 4079) showed the presence of the fengycin biosynthetic gene. The zwittermicin A gene was detected in B. mycoides S, Bacillus thuringiensis BS8, and B. amyloliquefaciens BS6. Sequence analysis of purified PCR products revealed homology with corresponding genes from other Bacillus sp. in the GenBank database. Production of particular antibiotics in strains BS6, H-08-02, 3057, and 4079 was confirmed through matrix-assisted laser desorption ionization - time of flight - mass spectroscopy (MALDI-TOF-MS). This study revealed the equivalent capability of different Bacillus strains from various microhabitats to produce the above-mentioned antibiotics and highlights the possibility of using some strains as potential biocontrol agents under different microhabitats distant from their original habitat. Furthermore, it will enable researchers to develop rational strategies for the application of the antagonists and their metabolites within an agroecosystem. To the best of our knowledge, this is the first report of a B. mycoides strain that carries biosynthetic genes and produces fengycin and surfactin.