Toxin-antitoxin (TA) systems have been considered essential factors for bacterial survival. During our drug development program aimed against tuberculosis (TB), we discovered certain peptides that mimic the binding of the VapBC30 complex, leading to the arrest of bacterial cell growth and eventually cell death. Herein, we optimized these candidate peptides based on a hydrocarbon stapling strategy and performed biological in vitro evaluations. The V30-SP-8 peptide successfully penetrated Mycobacterium smegmatis cell membranes and exerted bactericidal activity at a minimum inhibitory concentration that inhibited 50% of the isolates (MIC50) <6.25 M. With the aid of structural and biochemical information for the VapBC30 TA system from M. tuberculosis, we suggest potential antimicrobial agents that could provide a platform to establish a novel antibacterial strategy. Reflecting the limited number of therapeutic agents targeting TA systems, we believe that this study not only provides chemical tools for exploring the biological events relevant to TA systems but also opens a new gateway toward TB drug discovery.