Amphibian skin secretion has been an important source of broad-spectrum and membrane-targeting antimicrobial peptides, which promise to tackle the antibiotic resistance crisis.Callimedusa ecuatoriana from Ecuador is an example of an unexplored species, that can hold a library of novel chemical scaffolds with antibiotic action. In this study, we report a novel skin peptide (PTR-CE1) identified by molecular cloning of mRNA precursor. We demonstrated that it lacks of antimicrobial activity. So, using the natural sequence of PTR-CE1 as a template, we designed and synthesized two analogs (PTR-CE1a and PTR-CE1b). Both engineered peptides displayed high antibacterial activity, even against the ampicillin-resistant bacterial strains. While PTR-CE1b showed MIC values of 106.5-212.99 mM and less than 10% of damage to red blood cells at 3.02 mM, PTR-CE1a displayed a more potent broad-spectrum effect against all the tested microorganisms, with MIC values of 3.02-12.06 mM, and low hemolytic properties at 6.66 mM. This study highlights the role of the secondary structure for antimicrobial activity and shows how inactive peptides can be useful as a template for the generation of new molecules with high activity and low toxicity.