We have investigated the effects of supercritical carbon dioxide (SC-CO2) modified with water as a cosolvent on the inactivation of Penicillium oxalicum spores inoculated on barley grains and also on the germination yield of the SC-CO2-treated barley grains. After SC-CO2 treatment at different conditions of temperature, cosolvent (water) content and treatment times, the number of colony-forming units (cfus) of fungal spores and the germination of SC-CO2-treated barley grains were analyzed. Among the SC-CO2 treatment parameters, the cosolvent content was the most significant factor affecting the inactivation yields of P.oxalicum spores. When the pressure of SC-CO2 was fixed at 10MPa, the optimal conditions obtained by ridge analysis of response surface methodology were cosolvent content of 231μL, temperature of 44C and treatment duration of 12min, which resulted in a 6.8 log10 reduction of cfu. However, the germination yield of barley grains decreased significantly by treating with SC-CO2 modified with water. For example, the addition of only 300μL water in the SC-CO2 treatment reduced the germination yield from 65.4% to 1.4% at the same SC-CO2 treatment conditions (40C, 10MPa, and 20min). Practical Applications: This study demonstrated the lethal effect of modified supercritical carbon dioxide (SC-CO2) against plant pathogenic fungal spores in barley grains. Water cosolvent showed significant synergism in inactivating fungal spores with SC-CO2. Treatment using SC-CO2 modified with water cosolvent could be applied in the sterilization step for preservation of barley grains as an alternate for chemical or thermal processes.