The use of gaseous ozone as a fungicide to preserve stored barley was studied. The effects of the following operating parameters on the fungicidal efficacy of ozone were examined: 1) the applied ozone dose, 2) ozonation time, 3) water activity of barley, and 4) temperature of barley. The effect of ozonation on germination of barley was also investigated. The experimental results showed that ozone was very effective in inactivation of fungi associated with the barley regardless of whether the fungi were in the forms of spores or mycelia. However, the mycelia were less resistant to ozone. With 5 minutes of ozonation, 96% of inactivation were achieved for spores as well as for mixtures of spores and small amount of mycelia by applying 0.16 and 0.10 mg of ozone/(g barley) x min, respectively. In addition, for sealed storage silos, inactivation of fungi continued when the ozone-containing gas was held inside the silos following a continuous ozone supply. The experimental results also revealed that increases in water activity and temperature of barley enhanced the fungicidal efficacy of ozone. Results of this study also indicated that the inactivation processes could be controlled by simply monitoring the exit ozone from the reactor instead of performing the time-consuming microbial examination. This finding would make the application of ozone in the preservation of cereal grains easier, simpler, and more practically applicable. The experimental results demonstrated that although ozonation above certain strength may reduce barley germination, inactivation of fungi was achieved with ozonation strengths far below the critical point.