Amphibian skin secretions constitute an important source of molecules for antimicrobial drug research in order to combat the increasing resistance of pathogens to conventional antibiotics. Among the various types of substances secreted by the dermal granular amphibian glands, there is a wide range of peptides and proteins, often displaying potent antimicrobial activities and providing an effective defense system against parasite infection. In the present work, we report the NMR solution structure and the biological activity of a cationic 14-residue amphiphilic alpha-helical polypeptide named Hylaseptin P1 (HSP1), isolated from the skin secretion of the hylid frog Hyla punctata. The peptide antimicrobial activity was verified against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, whereas no significant lytic effect was detected toward red or white blood cells.