Pine wilt disease caused by Bursaphelenchus xylophilus is an extremely serious issue worldwide. Trichoderma asperellum is used as a biocontrol agent due to its efficient inhibition of various pathogens. Three solvents, methanol, petroleum ether, and acetone, were used for extracting the fermentation broth of T. asperellum. All three extracts displayed a significant difference in nematocidal efficacy of more than 70% at 72 hours. Water-soluble natural products or polyphenolic compounds exhibited the greatest nematocidal ability after 24 hours of treatment with methanol. However, lipophilic substances exhibit long-lasting nematocidal ability. During this process, the antioxidant enzyme activities of T. asperellum significantly increased, including those of catalase, superoxide dismutase, and peroxidase. The interaction between T. asperellum and B. xylophilus induces the synthesis of glycoside hydrolase family 18 (chitinases) and peptidase S8, potential subtilisin, which dissolve the body wall of the nematodes. Fatty acid metabolism was significantly increased, and a defensive response against substances secreted by the nematode was produced. Antioxidant reaction mechanisms and detoxification metabolism were enriched and included a large number of upregulated DEGs, including GST, CYP450, LysM, and RTA1. The dual and toxic mechanisms exhibited by T. asperellum during its response to B. xylophilus involve multiple pathways and genes that are collectively regulated. This is the first study to investigate the effects of T. asperellum on B. xylophilus. This study provides a theoretical basis for the potential use of T. asperellum as a biocontrol agent against B. xylophilus. Display omitted •All three extracts had a significant difference in nematicidal efficacy of more than 70%.•T. asperellum increased antioxidant enzyme activities including CAT, SOD, and POD.•Detoxification metabolism was enriched, including GST, CYP450, LysM, and RTA1.•Chitinases, and subtilisin dissolved the body wall of the nematodes.•In the network, three transporter and four oxidases in highly connected genes.