Eutrema japonicum is a perennial herb belonging to the Eutrema genus in the crucifer family. In recent years, numerous substances with notable pharmacological activities have been successfully isolated from E. japonicum. Despite significant advancements in related research, the efficacy of the rhizome extract of E. japonicum against root-knot nematodes remains unknown. In this study, the rhizome extract of E. japonicum was used as raw material to demonstrate the inhibitory and nematicidal effects of the extract on Meloidogyne enterolobii. The results showed that the LC₅₀ of the E. japonicum rhizome extract on second-stage juveniles (J2s) was 69.590 mg/mL and 22.336 mg/mL at 24 h and 48 h after treatment, respectively. The mortality rate of J2s reached 88.93% at 48 h post-treatment when the concentration was 200 mg/mL, and the inhibition rate of single-egg hatching reached 88.14%. This study analyzed the chemical composition of the ethanol extract of E. japonicum, and 10 organosulfur compounds and lipid compounds with insecticidal and antibacterial effects were preliminarily screened out. Among them, sec-butyl isothiocyanate and geraniol were further investigated for their nematicidal activity, demonstrating high efficacy against M. enterolobii. Moreover, we conducted network pharmacology analysis and RT-qPCR analysis to predict the potential inhibitory mechanisms of sec-butyl isothiocyanate and geraniol on M. enterolobii. These findings offer a scientific foundation and theoretical framework for utilizing E.japonicum as a potential raw material for developing novel natural plant nematicides.