The in vitro activity of four polycationic 9-mer peptides derived from insect defensins alone and combined with clinically used antibiotics was investigated against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. These bacteria were more susceptible to the D-9-mer peptides (diastereomer) composed of only D-amino acids (MIC range: 20-100μg/ml) than to the L-9-mer peptide composed of only L-amino acids (MIC range: 400-800μg/ml). Furthermore, D-9-mer peptides exhibited synergic and additive effects in combination with antibiotics. Interestingly, an increase in bacterial resistance to antibiotics was observed whereas resistance to D-9-mer peptides was not observed, even when bacteria were cultured in medium containing these antimicrobial peptides and antibiotics for several hundred generations (MRSA: 200 generations and P. aeruginosa: 1000 generations). Moreover, one D-9-mer peptide showed continuous synergy in combination with piperacillin against P. aeruginosa during cultivation. These results suggest the potential use of these D-9-mer peptides in topical or systemic therapy for nosocomial infections.