To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin‐1 (PG‐1), an 18‐residue cationic peptide with a broad‐spectrum antimicrobial activity. These cyclic protegrins bear end‐to‐end peptide bonds together with varying numbers (zero to three) of cross‐strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low‐ and high‐salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram‐positive and Gram‐negative bacteria, fungi and one tested virus, HIV‐1. Compared to PG‐1, the cyclic tricystine ccPG 3 displayed approximately a 10‐fold decrease in hemolytic activity against human cells and 6‐ to 30‐fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [ΔSS]cPG 8, a cyclic protegrin with no disulfide bond, and [ΔCys6,15]cPG 5, a cyclic mimic of PG‐1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG‐1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of β‐strand structures in water/trifluoroethanol or phosphate‐buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity.