The increasing prevalence of antimicrobial resistance underscores the urgent need for alternative treatments. Antimicrobial peptides (AMPs) are considered viable substitutes for conventional antibiotics due to their broad-spectrum activity and unique mechanisms of action. AMPs are an integral component of the innate immune system in invertebrates. Mastoparan-S, a 14-amino acid AMP originally identified in the praying mantis Sphodromantis viridis reportedly exhibits antimicrobial activity against gram-positive and gram-negative bacteria, as well as fungi. In this study, the antibacterial activity and mechanism of action of chemically-synthesized mastoparan-S are evaluated against Staphylococcus aureus and Pseudomonas aeruginosa. Mastoparan-S exhibits potent antibacterial activity against S. aureus and P. aeruginosa, without inducing cytotoxicity or hemolytic activity in RAW264.7 or HaCaT cells. Mastoparan-S effectively inhibits biofilm formation by S. aureus and P. aeruginosa. Mechanistically, mastopran-S kills bacteria by disrupting the bacterial membrane. Collectively, these findings suggest that mastoparan-S is a potential therapeutic agent for treating S. aureus and P. aeruginosa infections.