In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin-Ra and temporin-Rb, were identified and purified using RP-HPLC. Temporin-Ra and temporin-Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram-negative and gram-positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin-Rb and 1585.1 Da for temporin-Ra. Human red blood cells tolerated well exposure to temporin-Ra and temporin-Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide-spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.