The natural antimicrobial cationic peptide protegrin‐1 displays a broad spectrum of antimicrobial activity and rapidly kills pathogens by interacting with their cell membrane. We investigated the structure–activity relationships of three protegrin‐1 analogues: IB‐367 (RGGLCYCRGRFCVCVGR‐NH2), BM‐1 (RGLCYCRGRFCVCVG‐NH2) and BM‐2 (RGLCYRPRFVCVG‐NH2). Our antimicrobial and antifungal activity studies of these peptides showed that BM‐1 was much more active than IB‐367 against Gram‐positive bacteria and fungi, whereas BM‐2 was inactive. The BM‐1 peptide showed fourfold reduced haemolysis relative to IB‐367, an additional advantage of this peptide. In addition, BM‐1 was about 15% cheaper than IB‐367 to synthesize. The absence of two cysteine residues in the BM‐2 sequence could be the main reason for its unstable conformation and antimicrobial inactivity. The solution structures of these peptides were determined in dimethyl sulphoxide using two‐dimensional NMR and restrained molecular dynamics calculations. IB‐367 and BM‐1 formed short, antiparallel, β‐hairpin structures connected by a type II′β‐turn. The shorter, inactive BM‐2 analogue exhibited major and minor conformations (predominantly unordered) in the NMR spectra and was much more flexible.