We previously proved that by changing the configuration of only two amino acids in Esc(1–21), with the corresponding d-enantiomers, the resulting diastereomer Esc(1–21)-1c had a (i) significantly higher biostability, (ii) lower cytotoxicity, (iii) better antibiofilm activity, (iv) stronger ability in killing Pseudomonas internalized into bronchial cells expressing a copy of functional or mutated CF-transmembrane conductance regulator (AF508 CFTR) and (v) a more pronounced wound-healing activity in monolayers of epithelial cells29. All these findings contributed to make Esc(1–21)-1c a very attractive candidate for the development of new drugs against Pseudomonas-induced lung infections. Nevertheless, no evidences on its in vivo antimicrobial efficacy were previously reported. Here, for the first time we demonstrated a remarkable in vivo antipseudomonal activity of the diastereomer Esc(1–21)-1c after a single local instillation in the lungs, with a higher potency than the all-L parent peptide and the widely used and characterized human AMP LL-37. Our in vivo studies underlined that 4 hours peptide treatment is not an ideal time for an optimal evaluation of the in vivo antimicrobial effectiveness of esculentin-derived AMPs, which presumably relies on a direct bactericidal activity of the peptide. However, at this stage we cannot exclude that the respiratory epithelial cell-mediated immune modulation could also contribute to the overall host antimicrobial activity. The bacterial killing activity by Esc(1–21) and Esc(1–21)-1c would prevent inhaled/inoculated bacteria from propagate in mouse lung. Nonetheless, the bacterial infection-induced host innate immunity may be able to provide additional killing mechanism to control the severity of bacterial infection and to reduce inflammatory response. Importantly, we have found out that a single intra-tracheal instillation of Esc(1–21)-1c, at a very low peptide dosage i.e. 20 μM (0.1 mg/kg), is sufficient to cause approximately 2-log reduction in the lung Pseudomonas burden, within 24 hours from bacterial challenge. The data suggest that the diastereomer Esc(1–21)-1c likely has a sustained residence time in the lungs, due to its higher biostability and resistance to proteolytic degradation than Esc(1–21) and LL-37, according to our previously published data28, 29. Thus, it would continue to display microbicidal effects at longer-term. In comparison, the in vivo antipseudomonal activity of Esc(1–21)-1c resulted to be similar in bacterial reduction to that of colistin, which is extensively used in clinical practice43. Note however that colistin is a peptide which is active only against Gram-negative bacteria and it easily induces microbial resistance31, 44, 45, most likely because of modifications to the phosphate groups of lipid A and core oligosaccharide moieties of LPS, weakening colistin binding to it. Yet, colistin resistance in humans without prior exposure to this peptide has been recently disclosed; and this is an important concern to public health46.