This study investigated the antifungal activity and possible mechanisms of an extract from Lysurus mokusin(L134) against Pestalotiopsis neglecta.The effect of Lysurus mokusin(L134) on the enzyme activity, membrane permeability, and cell wall integrity of P. neglectawas assayed. Besides, we conducted a gas chromatography/mass spectrometry (GC–MS) to identify the individual compounds in Lysurus mokusin(L134). Pinus sylvestrisvar. mongolicaLitv was used as the study object to evaluate the prevention and treatment effect of Lysurus mokusinL134 extract against P. neglecta(black spot needle blight).The results showed that Lysurus mokusin(L134) has a potent inhibitory effect against P. neglectamycelial growth, and the inhibition rate is positively correlated with the concentration of Lysurus mokusin(L134). Besides, Lysurus mokusin(L134) can effectively inhibit spore germination in P. neglecta. Scanning electron microscopy (SEM) showed that after Lysurus mokusin(L134) treatment, the mycelia assumed an inward concave shape, became distorted, atrophied, and swollen. Evaluation of the antifungal mechanism indicated that Lysurus mokusin(L134) reduces cell wall integrity and increases cell membrane permeability. Furthermore, the effect of biocontrol revealed that L134 could effectively control black spot needle blight of Pinus sylvestrisvar. mongolica. GC–MS analysis of the Lysurus mokusin(L134) fraction obtained from n-butanol revealed d-glucitol,hexaacetate, l-( +)-rhamnose,aldononitrile,tetraacetate are the major compounds. The present study is the first report that these compounds have fungistatic activity. Overall, this study suggests that Lysurus mokusin(L134) extract can potentially be used as a fungicide for the sustainable management of plant diseases.