Antimicrobial peptides (AMPs) can target bacterial membranes and kill bacteria through membrane structural damage and cytoplasmic leakage. A group of surfactant-like cationic AMPs was developed from substitutions to selective amino acids in the general formula of G(IIKK)3I-NH2, (called G3, a de novo AMP), to explore the correlation between AMP hydrophobicity and bioactivity. A threshold surface pressure over 12 mN/m was required to cause measurable antimicrobial activity and this corresponded to a critical AMP concentration. Greater surface activity exhibited stronger antimicrobial activity but had the drawback of worsening hemolytic activity. Small unilamellar vesicles (SUVs) with specific lipid compositions were used to model bacterial and host mammalian cell membranes by mimicking the main structural determinants of the charge and composition. Leakage from the SUVs of encapsulated carboxyfluorescein measured by fluorescence spectroscopy indicated a negative correlation between hydrophobicity and model membrane selectivity, consistent with measurements of the zeta potential that demonstrated the extent of AMP binding onto model SUV lipid bilayers. Experiments with model lipid membranes thus explained the trend of minimum inhibitory concentrations and selectivity measured from real cell systems and demonstrated the dominant influence of hydrophobicity. This work provides useful guidance for the improvement of the potency of AMPs via structural design, whilst taking due consideration of cytotoxicity.