As the global occurrence of drug-resistant antimicrobial infections increase at an alarming rate, exacerbated by COVID-19, and the forefront of a ‘post antibiotic’ era approaches, antimicrobial peptides (AMPs) have been thrust into the spotlight.1 These host defense molecules found throughout all walks of life have caught the eye of researchers worldwide for their unique broad-spectrum potency to a variety of pathogens.2 Disulfide rich peptides (DRPs) are unique within AMPs, namely due to their highly defined secondary structures and multicyclic nature. We recently reported the first total synthesis of a class leading antimicrobial peptide, capitellacin, via, a now standardized, uniquely facile process incorporating manual flow-SPPS and orthogonally directed on-resin regioselective sequential disulfide formation affected by iodine.3-6 Furthermore, our optimized synthesis presented considerations for introducing multiple Cys residues under elevated flow-SPPS temperatures applicable for a variety of DRPs, with the added caveat of avoiding solution phase manipulations or aqueous work-ups employed during previous similar β-hairpin syntheses.6 Building upon our initial synthesis we designed, synthesized and investigated a library of five analogues to probe capitellacin’s surrounding chemical space, aiming to shed light on its presently obscure mechanism of action, and investigate the importance of secondary structure for activity. Our library incorporates analogues with variable cyclic structures rather than single amino acid residue replacements, a methodology previously shown to be successful in tuning the therapeutic potential of similarly designed β-hairpin analogues, including arenicin-III and tachyplesin-I.7,8 Alongside structure analysis by NMR and circular dichroism their antimicrobial activity against Gram-negative and Gram-positive bacteria was evaluated. Together the biological and structural data from our analogues provided important insights not only into the areas of possible AMP development for capitellacin, but highlighted the importance of the structural integrity of the β-hairpin for the antibacterial, and furthermore therapeutic potential of such DRPs.