Fusarium is a large genus of filamentous fungi widely distributed around the world and often associated with contamination of mycotoxins, representing one of the most prevalent sources of food contamination, with strong, acute or chronic negative impacts on human health. The complete elimination of fungi-contaminated commodities may not be indeed achievable, but a reduction in these is essential for final product quality as well as for consumer health. For this reason, the objective of this study was to evaluate the efficacy of the ozone (O-3) treatment in Fusarium species and, in addition, to investigate the effect of O-3 in malting barley seed germination. The O-3 gas was applied to pilot cylinders, divided into control, without O-3 gas application, and treated groups, with concentrations of 40 and 60 mg/kg. These were exposed for 30, 60, 120 and 180 min. The species that demonstrated the greatest reduction after the O-3 treatment was Fusarium poae, represented by 93.3%, followed by Fusarium graminearum, with 92.6%. Germination of the malting barley seed was not affected.