D-Alaninepeptidase purified from Bacillus amyloliquefaciene CMB01 caused a reduction of survival of Proteus vulgaris, Klebsiella oxytoca, and Staphylococcus aureus placed under the osmotic pressure. D-Alaninepeptidase caused an increase of susceptibility of bacteria to antibiotics. An increased number of malformed cells in bacterial groups exposed to D-alaninepeptidase was observed by scanning electron microscopy. These data suggested that bacterial cells exposed to D-alaninepeptidase resulted in an increase of vulnerability of bacterial cells toward environmental stress, such as osmotic pressure and antibiotic substances.