Abstract: Soybean root rot is an important soil-borne disease affecting soybean yield in China, and Fusarium oxysporum is its main causal agent. To determine the control potential of the novel sterol demethylation inhibitor (DMI) mefentrifluconazole against this disease, this study evaluated the pathogenicity of three F. oxysporum isolates, clarified the inhibitory activities of mefentrifluconazole mycelial growth, microconidiation, and germ-tube elongation of this pathogen. Furthermore, the protective and curative effects against soybean root rot were assessed, and the impact of mefentrifluconazole on ergosterol biosynthesis in F. oxysporum was investigated. The results showed that all three strains of the pathogen could cause disease in soybeans, with disease indices ranging from 26.98 to 49.21. Mefentrifluconazole exhibited good inhibitory effects on different developmental stages of F. oxysporum, with the best inhibition observed against mycelial growth, yielding EC₅₀ values of 0.52-0.54 μg/mL. Furthermore, mefentrifluconazole exhibits both protective and curative activities against root rot in soybean and significantly suppresses ergosterol biosynthesis in F. oxysporum. These findings clarify the inhibitory activity of mefentrifluconazole against the soybean root rot pathogen, which can provide a reference for the scientific application of mefentrifluconazole and other DMI fungicides in the control of soybean root rot.