Aphelenchoides besseyi and Ditylenchus destructor can cause serious problems for a number of important agricultural crops and vegetables. In this study, the ethanol extract of Stellera chamaejasme L. roots showed strong nematicidal activity against Aphelenchoides besseyi and Ditylenchus destructor. By using a bioactivity-driven fractionation, three biflavonoids were isolated from the extract and their structures were identified by mass and nuclear magnetic resonance spectral data. Nematicidal activity bioassays revealed that isoneochamaejasmin A had the strongest nematicidal activity against A. besseyi and D. destructor with LC50 values of 2.32 and 0.18 mM at 72 h, respectively. Chamaejasmenin B displayed weaker nematicidal activity against A. besseyi with an LC50 value of 3.94 mM at 72 h. Neochamaejasmin B induced the lowest mortality against D. destructor with an LC50  values of 15.6 mM at 72 h. These results suggested that the kind and position of substitutions and the relative configuration of 2-H/3-H and 2”-H/3”-H could be considered as important factors responsible for the nematicidal activity of these purified C-3/C-3″ biflavonoids.