This work is focused on the use of supercritical carbon dioxide to inactivate the pathogenic Escherichia coli. For this purpose, the experimental design methodology was employed to evaluate the effects of pressure (8–28 MPa), depressurization rate (1–11 MPa min−1) and pressure cycling (1–5 cycles) on the microorganism inactivation using the static-synthetic method in a variable-volume reactor. The number of pressure cycles and system pressure showed a significant influence on E. coli inactivation with supercritical CO2, indicating that the increase in the number of pressure cycles and system pressure improve inactivation efficiency. Microbial inactivation followed first order reaction kinetics, where the rates increased with increasing pressure from 8 to 16 MPa. The decimal reduction times (D) ranged from 1.03 to 5.35 min. The pressure dependence of the E. coli specific inactivation rates can be described by the z value, which was found to be 11.4. The results reported here may be useful to provide an effective non-thermal sterilization of foods in pilot/industrial scale.