The inactivation of spores of Penicillium oxalicum by supercritical carbon dioxide (SC-CO2) was optimized by response surface methodology. The optimal inactivation conditions of 16.8 MPa, 49°C, and 20 min were determined using ridge analysis, at which the predicted and experimental log10 reductions were obtained as 5.74 and 6.12, respectively. The synergistic effect of a cosolvent (ethanol), which was used to modify SC-CO2, on the inactivation of the fungal spores was investigated. At less severe conditions of 10 MPa and 40°C, P. oxalicum spores of 10(7) CFU/ml were completely inactivated within 45 min by SC-CO2 modified with ethanol.