Rice blast, caused by Magnaporthe grisea, is one of the most destructive diseases of rice worldwide. Mefentrifluconazole, a novel isopropanol triazole fungicide, exhibits excellent bioactivity to a wide range of fungi. However, its antifungal activity against M. grisea is not well studied. In this study, the sensitivity of 103 M. oryzae strains to mefentrifluconazole was determined by mycelial growth rate method, and the effect of mefentrifluconazole on the biochemical and physiological characteristers was examined. The results indicated that mefentrifluconazole exhibited excellent inhibitory activity against M. oryzae with EC50 values of 103 M. oryzae strains tested ranging from 0.363 to 1.576 μg/mL, and the average EC50 value of 0.958 ± 0.298 μg/mL. After mefentrifluconazole treatment, the apical branches of M. oryzae mycelia increased and the mycelia became distorted, twisted and ruptured. In addition, mefentrifluconazole increased relative conductivity, disrupted cell wall and membrane integrity, and significantly reduced ergosterol content. The expression levels of demethylation inhibitor resistance-associated genes Cyp51A, ABC6 and MFS1 were all significantly upregulated, suggesting an adaption response to mefentrifluconazole of M. oryzae. Furthermore, the results of pot experiment showed that mefentrifluconazole provided >70% control efficacy against rice blast at 300 μg/mL, surpassing the performance of carbendazim, prochloraz and azoxystrobin at the same concentration. Overall, this study demonstrated the strong inhibitory activity of mefentrifluconazole against M. oryzae and the excellent efficacy in controlling rice blast, thus providing new sights into rice blast control as well as the practical application of mefentrifluconazole.