Selectivity is a key requirement for membrane-active antimicrobials to be viable in therapeutic contexts. Therefore, the rational design or suitable selection of new compounds requires adequate mechanistic understanding of peptide selectivity. In this study, we compare two similar cyclic peptides that differ only in the arrangement of their three hydrophobic tryptophan (W) and three positively charged arginine (R) residues, yet exhibit different selectivities. This family of peptides has previously been shown to target the cytoplasmic membrane of bacteria, but not to act directly by membrane permeabilization. We have systematically studied and compared the interactions of the two peptides with zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylglycerol/phosphatidylethanolamine (PG/PE) model membranes using various biophysical methods to elucidate the mechanism of the selectivity. Like many antimicrobial peptides, the cyclic, cationic hexapeptides investigated here bind more efficiently to negatively charged membranes than to zwitterionic ones. Consequently, the two peptides induce vesicle leakage, changes in lipid packing, vesicle aggregation, and vesicle fusion predominantly in binary, negatively charged PG/PE membranes. The peptide with the larger hydrophobic molecular surface (three adjacent W residues) causes all these investigated effects more efficiently. In particular, it induces leakage by asymmetry stress and/or leaky fusion in zwitterionic and charged membranes, which may contribute to high activity but reduces selectivity. The unselective type of leakage appears to be driven by the more pronounced insertion into the lipid layer, facilitated by the larger hydrophobic surface of the peptide. Therefore, avoiding local accumulation of hydrophobic residues might improve the selectivity of future membrane-active compounds.