This work evaluates the inactivation of Listeria monocytogenes through the use of supercritical carbon dioxide. For this purpose, the experimental design methodology was employed as a tool to assess the effects of temperature, depressurization rate, pressure cycling and the mass ratio of cell suspension to CO2. It was observed that the depressurization rate and mass of cell suspension to CO2 ratio were statistically significant and inactivation kinetics was verified to be of first-order. It was also observed that the inactivation increased with increasing depressurization rates in the range of 6–12 MPa min−1, keeping nearly constant above the upper value. The decimal reduction times ranged from 15.38 to 20.41 min. Results obtained in this work may be quite useful to the food industry committed to microorganism inactivation using the innovative supercritical carbon dioxide technology.