Due to their capacity to produce antimicrobial peptides that can prevent the growth of diseases, manyBacillusspp. are beneficial to plants. In this study, we looked into the antagonistic activity of theB. pumilus3-19 strain and its derivatives following targeted genome editing. Two peptide genes with antibacterial action, bacilysin (bac) and bacteriocin (bact), and thesigF gene, which encodes the sigma factor of sporulation, were specifically inactivated using the CRISPR-Cas9 system in the genome ofB. pumilus3-19. Antibacterial activity againstB. cereusandPantoea brenneridecreased as a result of the inactivation of target genes in theB. pumilus3-19 genome, with a noticeable effect against bacilysin. The growth dynamics of the culture changed when thebac,bact, andsigF genes were inactivated, and the altered strains had less proteolytic activity. An asporogenic mutant ofB. pumilus3-19 was obtained by inactivating thesigF gene. It has been proven that bacilysin plays a unique part in the development ofB. pumilus3-19’s antagonistic action against soil microorganisms.