<jats:title>Abstract</jats:title><jats:p>Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. The present study investigates the antimicrobial activity of new, rationally-designed derivatives of a short α-helical peptide, RR. From the peptides designed, RR4 and its D-enantiomer, D-RR4, emerged as the most potent analogues with a more than 32-fold improvement in antimicrobial activity observed against multidrug-resistant strains of <jats:italic>Pseudomonas aeruginosa and Acinetobacter baumannii</jats:italic>. Remarkably, D-RR4 demonstrated potent activity against colistin-resistant strains of <jats:italic>P. aeruginosa</jats:italic> (isolated from cystic fibrosis patients) indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D-RR4 was more capable of disrupting <jats:italic>P. aeruginosa</jats:italic> and <jats:italic>A. baumannii</jats:italic> biofilms when compared to conventional antibiotics. Of note, D-RR4 was able to bind to lipopolysaccharide to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected <jats:italic>Caenorhabditis elegans</jats:italic> from lethal infections of <jats:italic>P</jats:italic>. <jats:italic>aeruginosa</jats:italic> and <jats:italic>A</jats:italic>. <jats:italic>baumannii</jats:italic> and enhanced the activity of colistin <jats:italic>in vivo</jats:italic> against colistin-resistant <jats:italic>P</jats:italic>. <jats:italic>aeruginosa</jats:italic>.</jats:p>