Abstract: Corm rot is the most serious disease on saffron, a Chinese medical herb belonging to "New Zhe Ba Wei". The sensitivity of 82 isolates of the pathogenic Fusarium oxysporum to a novel sterol demethylase inhibitor (DMI) mefentrifluconazole was determined using the mycelial growth rate method and the effects of mefentrifluconazole on the growth, sporulation, spore germination, and cell membrane permeability of F. oxysporum were investigated. The most and least sensitive strains in the population were used to determine the biological activity of mefentrifluconazole against F. oxysporum and the protective and curative efficacy were determined. The results showed that the EC₅₀ values of mefentrifluconazole inhibiting mycelial growth of F. oxysporum ranged from 0.182 μg/mL to 2.491 μg/mL, and the average EC₅₀ value was (0.838 ± 0.438) μg/mL. The sensitivity frequency distribution conforms to normal distribution. Mefentrifluconazole significantly decreased mycelial growth and sporulation of F. oxysporum. The conductivity test of mycelia demonstrated that mefentrifluconazole damaged cell membrane of F. oxysporum resulting in electrolyte leakage. In addition, the protective efficacy of mefentrifluconazole against saffron corm rot was superior to the curative efficacy. These results provide a theoretical basis for the effective control of saffron corm rot in production.