【Objective】Injecting agents into pine trees to prevent pine wilt disease is an effective control measure. This study aimed to develop more efficient and fast-delivery agents by combining two different drugs to create a novel microemulsion. The virulence activity of this combined (3% fluopyram-5% emamectin benzoate) microemulsion against Bursaphelenchus xylophilus and its potential for controlling pine wilt disease were explored.【Method】The nematocidal effects of the newly developed combined microemulsion, along with two commonly used agents for pine wilt disease-5% emamectin benzoate microemulsion and 5% avermectin emulsifiable concentrate-were assessed using the insect immersion method. B. xylophilus was treated with lethal concentrations (LC₁₀ and LC₃₀) of each agent to determine their impact on nematode reproduction. Three agents were injected into the trunks of Pinus massoniana trees in the forest to observe agent absorption and tree sensitivity to successfully absorbed agents. After 90 days of injection, samples of the treated trees were collected to analyze the dynamic distribution of drug transport within the tree body. Additionally, 100 days after inoculation, the treated trees were artificially inoculated with B. xylophilus to observe and statistically analyze the control effects of each agent on pine wilt disease.【Result】The LC₅₀ values after 24 hours of treatment with the 3% fluopyram-5% emamectin benzoate microemulsion, 5% emamectin benzoate microemulsion, and 5% avermectin emulsifiable concentrate on pine wood nematodes were 2.644 0, 27.678 4, and 42.891 1 mg/L, respectively. Among the tested agents, the 3% fluopyram-5% emamectin benzoate microemulsion exhibited the strongest nematocidal activity. The population reproduction of nematodes treated with low concentrations of the agents was significantly lower than the control group. Seven days after injection, both the self-made combined microemulsion and 5% avermectin emulsifiable concentrate were fully absorbed by the tree, whereas absorption of the 5% emamectin benzoate microemulsion was more limited. After 180 days, the self-made combined microemulsion showed minimal sensitivity to the tree, with slight discoloration at the injection point, while the 5% avermectin emulsifiable concentrate caused significant discoloration of the xylem at the injection site. After 90 days, residual concentrations of all agents were detected at the top of the tree. The highest residual concentration of fluopyram (0.13 mg/kg) was found 0.5 m below the injection point in the combined 3% fluopyram-5% emamectin benzoate microemulsion, while emamectin benzoate had a residual concentration of 0.07 mg/kg, and 5% avermectin's residual concent ration had 0.1 mg/kg. The highest residual concentrations of all agents were observed 0.5 m above the injection point, with fluopyram in the combined microemulsion reaching 55.65 mg/kg, significantly higher than the other agents. Overall, the residual concentration of fluopyram was higher than that of emamectin benzoate in the combined microemulsion and higher than 5% avermectin emulsifiable concentrate. Inoculation with pine wood nematodes in mid-July showed that, 270 days after inoculation, the trees treated with the agents remained healthy, while untreated trees exhibited signs of infection 100 days post-inoculation. All agents effectively prevented the onset of pine wilt disease.【Conclusion】The toxicity analysis of the new self-made combined agent demonstrates its strong nematocidal effect, good absorbability after injection into P. massoniana, and mild sensitivity to the tree. It also exhibits excellent transport ability within the tree and provides effective prevention against pine wood nematode infection. Further optimization of the formula and large-scale application in forest management is recommended.