The aim of this study is to investigate the bactericidal effect of supercritical nitrous oxide (SC N(2)O) on both typical Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli while varying the operating conditions including the suspending medium (nutrient broth, phosphate buffered saline, phosphate buffer, phosphate buffer containing oleic acid), pressure, temperature, mixing intensity, and working volume (the ratio of the sample volume to the reactor volume). Approximately 7 log of both S. aureus and E. coli by SC N(2)O (10 MPa, 37°C, 10% working volume, and 600 rpm) were completely inactivated within 20 min, although S. aureus exhibited relatively low sensitivity to SC N(2)O compared to E. coli. The bactericidal efficiency was not adversely affected by varying the suspending medium and the presence of oleic acid enhanced the inactivation. The mixing intensity, which is presumed to be associated with the increasing frequency of bacteria cells in contact with SC N(2)O as a result of the fast mass transfer of N(2)O molecules into the water layer containing the microbe, significantly affected the bactericidal activity, which was also supported by the % volume expansion of the aqueous phase. On the other hand, the other parameters (pressure, temperature, and working volume) appeared to moderately affect the bactericidal efficiency as long as the SC condition was maintained. This study reports that SC N(2)O technology can be effectively employed as an alternative for SC CO(2) sterilization technique in order to control bacteria in food which contains nutrients or fat in highly phosphate buffered conditions.