The peptidomimetic approach has emerged as a powerful tool for overcoming the inherent limitations of natural antimicrobial peptides, where the therapeutic potential can be improved by increasing the selectivity and bioavailability. Restraining the conformational flexibility of a molecule may reduce the entropy loss upon its binding to the membrane. Experimental findings demonstrate that the cyclization of linear antimicrobial peptoids increases their bactericidal activity against Staphylococcus aureus while maintaining high hemolytic concentrations. Surface X-ray scattering shows that macrocyclic peptoids intercalate into Langmuir monolayers of anionic lipids with greater efficacy than for their linear analogues. It is suggested that cyclization may increase peptoid activity by allowing the macrocycle to better penetrate the bacterial cell membrane.