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Antimicrobial peptides (AMPs) are lead candidates for antibacterial drug development, crucial amid rising antimicrobial resistance. A fundamental challenge is the development of antimicrobials effective against multidrug-resistant Gram-negative bacteria, largely due to the complexity of the outer membrane. In a previous study we showed the broad-spectrum and potent activity of Opis16a, a novel scorpion venom-derived AMP, against a large panel of Gram-negative bacteria, including drug-resistant strains, all while maintaining minimal toxicity to mammalian cells. However, the exact mechanism by which Opis16a acts even against antibiotic-resistant pathogens has not yet been elucidated. Here we show Opis16a effectively penetrates the intricate outer membrane of Gram-negative bacteria, leading to rapid bacterial cell death via cytoplasmic membrane disruption. In vitro screening confirms the efficacy of this peptide with activity against Gram-negative bacteria and potent selectivity toward most of these bacteria over human HaCat and HepG2 cell lines. Mechanistic studies in Escherichia coli and Acinetobacter baumannii reveal that Opis16a binds to lipopolysaccharides, leading to the rapid destabilisation of the outer membrane and subsequent depolarisation and permeabilisation of the inner cytoplasmic membrane within minutes. The strong antibacterial activity of Opis16a (MIC: 8 µg/mL), combined with its selectivity for bacterial over mammalian cells, effective membrane penetration of A. baumannii, and sustained activity in serum, translates into successful therapy in an in vivo Galleria mellonella model of gentamicin-resistant A. baumannii wound infection. Opis16a treatment nearly completely rescues all larvae from infection, demonstrating efficacy comparable to that of polymyxin B. This study identifies Opis16a as a novel membrane destabilising AMP with promising in vitro and in vivo activity against Gram-negative bacteria, with potential to be further developed for topical applications.