Skin secretions are known as a highly-complex mixture of abundant and diverse bioactive molecules and its study has attracted increasing attention over recent years. Phylloseptin is a unique family of antimicrobial peptides which have been only isolated from frogs of the Phyllomedusinae subfamily. Here, three novel peptide precursors were successfully cloned from a cDNA library, which was constructed from the skin secretion of Phyllomedusa burmeisteri, as pair of primers (one nested universal primer and a designed degenerate sense primer) were employed for "shotgun" cloning. The encoded mature peptides were validated by MS/MS sequencing, and subsequently termed as Phylloseptin-PBa1, -PBa2 and -PBa3. Phylloseptin-PBa1 and -PBa2 were demonstrated to possess potent antimicrobial activities against Gram-positive bacteria and yeast, as well as broad-spectrum anticancer activities, while they possess varying haemolytic activity at the effective concentration. In contrast, Phylloseptin-PBa3 was found to exhibit a strong haemolytic activity even though it was only found to possess a weak antimicrobial activity and inconspicuous anticancer activity. (C) 2018 Elsevier Inc. All rights reserved.