Over many decades, naturally-occurring substances have been considered as an integral part of the treatment of human diseases. Skin secretions from amphibians play an important role for sourcing such agents with therapeutic potential. Amongst all types of substances discovered from amphibian skin secretion, peptides represent a large family of bioactive molecules with distinctive biological functions. To date, more than 2000 host defence peptides have been studied and many of them possess antimicrobial, antifungal or anticancer properties. In the present study, bioactive peptides have been isolated from skin secretions of four amphibians, the green tree frog Litoria caerulea, the white-lipped tree frog Litoria infrafrenata, the green and golden bell frog Litoria aurea and the European edible frog Rana esculenta. Amongst three bioactive peptides, three belong to the neuromedin U family and the other is a brevinin analogue. By using genomic and proteomic techniques, biosynthetic precursor-encoding cDNAs from each species were selectively cloned. The chemical structures of mature peptides was confirmed by MS/MS fragmentation sequencing and reverse phase HPLC. Biological functions of their synthetic replicates were screened in functional assays, including smooth muscle preparations and minimum inhibitory concentration assays for determining effects on microorganism growth.