Taking into account the attractive properties of the two esculentin-derived peptides, we first evaluated their outcome on the bronchial epithelium permeability followed by their therapeutic efficacy in murine models of acute Pseudomonas lung infections, upon intra-tracheal instillation. Their effects on the expression of inflammation associated genes were also investigated. Overall, the results of our experiments have pointed out a higher in vivo antipseudomonal activity of the diastereomer Esc(1–21)-1c than its all-l counterpart without provoking any noticeable inflammation or damage at the lung level. Importantly, our work is the first demonstration of a frog-skin derived AMP to significantly promote lung clearance of P. aeruginosa when administered in a single dose at 2 hours after infection. The designed diastereomer demonstrated a higher antibacterial activity than the mammalian AMP LL-37, as well as a comparable potency to that of the clinically used colistin peptide35, 36, whose antibacterial activity is exerted through binding to LPS, the major component of the outer membrane of Gram-negative bacteria.