The growing occurrence of bacterial resistance to conventional antibiotics has called for the development of new classes of antimicrobial agents. Antimicrobial peptides (AMPs) with broad antimicrobial spectrum derived from frog skin secretions have been demonstrated to be promising candidates for new antibiotic development. A proven rich source of these compounds are the skin secretions of the frogs in the Phyllomedusa genus. In this study, two novel phylloseptin peptides-phylloseptin-PTa and phylloseptin-PHa-were isolated from the skin secretions of the South American frogs, Phyllomedusa tarsius (P. tarsius) and Phyllomedusa hypochondrialis (P. hypochondrialis) through parallel transcriptomic and peptidomic studies. Replicates obtained by chemical synthesis were structurally analysed and shown to adopt an α-helix configuration in an amphiphilic environment. Both peptides demonstrated antimicrobial activities against planktonic Gram-positive bacteria strains, including Staphylococcus aureus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus , biofilms, as well as cytostatic effects on the non-small cell lung cancer cell line, NCI-H157, with relatively low haemolysis on horse erythrocytes and low cytotoxicity on the human microvascular endothelial cell line, HMEC-1. The discovery of phylloseptin peptides may further inspire the development of new types of antibiotics.