Skin plays a key role in the daily survival of amphibians. In the present study, six cDNAs encoding amphibian skin antimicrobial peptide precursors from the Chinese brown frog Rana chensinensis, were cloned and identified as preprobrevinin-1CEa, preprobrevinin-1CEb, preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, and preprochensinin-1. Preprotemporin-1CEa, CEb, and CEc are members of the temporin family, which are usually short, hydrophobic, and C-terminally α-amidated antimicrobial peptides. Preprobrevinin-1CEa and CEb were identified as members of the brevinin-1 family of antimicrobial peptides, because both peptides contain a “Rana box” that is responsible for forming C-terminal Cys-bridged cyclic heptapeptides. The nucleotide and deduced amino acid sequences of preprochensinin-1 were not similar to any known amphibian skin defensive peptides. Four bioactive peptides were chemically synthesized according to the deduced amino acid sequences of six prepropeptides from R. chensinensis skin, and their antimicrobial, cytotoxic, and haemolytic properties were evaluated. All of the synthesized peptides inhibited the growth of Gram-positive bacteria. Brevinin-1CEa showed a broad spectrum of antimicrobial activity. The novel amphibian skin peptide chensinin-1 was active against Bacillus cereus and Streptococcus lactis at a concentration of 11.6 µM, but did not inhibit the growth of MCF-7 and HeLa cells at 200 µM, and had no haemolytic activity at a concentration of 500 µM. Temporin-1CEa exhibited the greatest ability to inhibit the growth of MCF-7 cells. Its antimicrobial and cytotoxic activities may be due to its high degree of α-helical confirmation and amphipathic nature.