Brevinin-2 related peptide (B2RP; GIWDTIKSMG10KVFAGKILQN20L.NH2), first isolated from skin secretions of the mink frog Lithobates septentrionalis, shows broad-spectrum antimicrobial activity but its therapeutic potential is limited by moderate hemolytic activity. The peptide adopts an α-helical conformation in a membrane-mimetic solvent but amphipathicity is low. Increasing amphipathicity together with hydrophobicity by the substitutions Lys16→Leu and Lys16→Ala increased hemolytic activity approximately fivefold without increasing antimicrobial potency. The substitution Leu18→Lys increased both cationicity and amphipathicity but produced decreases in both antimicrobial potency and hemolytic activity. In contrast, increasing cationicity of B2RP without changing amphipathicity by the substitution Asp4→Lys resulted in a fourfold increase in potency against Escherichia coli [minimal inhibitory concentration (MIC) = 6 μm) and twofold increases in potency against Staphylococcus aureus (MIC = 12.5 μm) and Candida albicans (MIC = 6 μm) without changing significantly hemolytic activity against human erythrocytes (LC50 = 95 μm). The emergence of antibiotic-resistant strains of the Gram-negative bacterium Acinetobacter baumannii constitutes a serious risk to public health. B2RP (MIC = 3–6 μm) and [Lys4]B2RP (MIC = 1.5–3 μm) potently inhibited the growth of nosocomial isolates of multidrug-resistant Acinetobacter baumannii. Although the analogs [Lys4, Lys18]B2RP and [Lys4, Ala16, Lys18]B2RP showed reduced potency against Staphylococcus aureus, they retained activity against Acinetobacter baumannii (MIC = 3–6 μm) and had very low hemolytic activity (LC50 > 200 μm).