Non-thermal processes for the effective sterilization and inactivation of microorganisms are currently receiving a great deal of attention in food, pharmaceutical and other relevant industries. Supercritical carbon dioxide (SC-CO(2)) treatment is an alternative method of microbial inactivation that can be safely used in foods and bioactive materials at relatively low temperatures. However, to date, the inactivation of microbial cells by treatment with SC-CO(2) has only been evaluated using a conventional plating method. Therefore, it is difficult to quantitatively determine the damage to cells other than counting colony forming units and also to find the possible inactivation mechanism by SC-CO(2) treatment. For the first time in the area of SC-CO(2) inactivation of microorganisms, we analyzed the physiological status of SC-CO(2) treated Salmonella enterica serotype Typhimurium using flow cytometry and then compared the flow cytometric data to the survival rate obtained by the plating method. The results of these systemic analyses revealed that SC-CO(2) caused damage to various aspects of the cells, including the efflux pump and membrane integrity.