Ocellatins are peptides produced in the skins of frogs belonging to the genus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Barbour 1906 and Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from (ocellatin-1N, -2N, -3N, and -4N). While ocellatins-1I, -2I, and -1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH) was active against an antibiotic-resistant strain of and reference strains of , , , and (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 μM), and was the only peptide active against Gram-positive (MIC = 31.25 μM) and (MIC = 62.5 μM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC = 98 μM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens.