The studies of the effect of supercritical carbon dioxide (scCO2) pasteurization on solid food from fish origin are scarcely available. This study was intended to address that gap by investigating the effect of scCO2 on the reduction of muscle protease activity and its impact on physicochemical properties of the Nile tilapia. Tilapia were exposed to CO2 pressure at 70, 75, 80, 85, and 90 bar; temperature at 40 °C; and holding time for 15 min. This study discovered that 80 bar was the minimum pressure to achieve half residual activity of muscle protease and two logs reductions of microbial counts. The applications of 80 and 85 bar were found to achieve significant reduction of tilapia muscle protease activity while still maintained acceptable textural properties. Both 80 and 85 bar were found to be effective to inhibit softening development of tilapia fillet during 14 days of chilled storage. Eighty-five bar and 15 min CO2 pasteurization was considered as maximum level of CO2 pressure that tilapia could withstand without degrading its texture significantly.