Non-thermal sterilization and microbial inactivation processes are currently receiving much attention in food and pharmaceutical industries. In particular, since supercritical carbon dioxide (SC-CO2) treatment, which is conducted at relatively low temperatures, is considered to be a promising alternative method to replace thermal sterilization processes that cannot be safely used in foods and bioactive materials. Although SC-CO2 has been applied to many microorganisms, the inactivation of microbial cells by SC-CO2 has only been evaluated by using a conventional viable cell count such as a plating method, by which it is not possible to systematically elucidate the microbial cell inactivation process. Therefore, in this study the physiological status of SC-CO2 treated Salmonella enterica serotype Typhimurium was analyzed by using GC-MS analysis of fatty acids with principal component analysis and two-dimensional electrophoresis for protein profiling. From the results of these systemic analyses, it was revealed that SC-CO2 caused significant alterations to the profiles of fatty acids and proteins of the cells.