The pine wilt disease is caused by the pinewood nematode Bursaphelenchus xylophilus and it results in serious ecological and economic losses. Therefore, effective prevention and control methods for the pinewood nematode are urgently required. Bacillus thuringiensis (Bt), a widely used microbial insecticide, produces toxins that are toxic to several species of parasitic nematodes, however, its effects on B. xylophilus have not been determined. In this study, Cry5Ba3, App6Aa2, Cry12Aa1, Cry13Aa1, Cry14Aa1, Cry21Aa3, Cry21Fa1, Xpp55Aa1, and Cyt8Aa1 toxins’ nematocidal activity against B. xylophilus was evaluated, six toxins with high toxicity were identified: App6Aa2 (LC₅₀ = 49.71 μg/mL), Cry13Aa1 (LC₅₀ = 53.17 μg/mL), Cry12Aa1 (LC₅₀ = 58.88 μg/mL), Cry5Ba3 (LC₅₀ = 63.99 μg/mL), Xpp55Aa1 (LC₅₀ = 65.14 μg/mL), and Cyt8Aa1 (LC₅₀ = 96.50 μg/mL). The six toxins caused shrinkage and thinning of the intestinal cells, contraction of the intestine from the body wall, vacuolization, and degenerated appearance of the pinewood nematodes. The results of this study provide basic information to study the action mechanism of nematocidal toxins on the pinewood nematode and direction for the use of nematocidal toxins in the biological control of B. xylophilus.