Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept <i>in vivo</i> experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by ε-lysylation.