Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by 'de novo' mass spectrometry and grouped into Dermaseptins (DPh-1).