Abstract: Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus, is a devastating forest disease that poses a severe threat to pine ecosystems. This study aimed to enhance the nematicidal activity of Bacillus velezensis Pt-RP9 by optimizing its fermentation process, providing fundamental data for its application in the biocontrol of pine wilt disease. Nematicidal assays, ultrastructural observations, and lipid droplet staining were conducted to evaluate the efficacy and mode of action of Pt-RP9 fermentation products. In addition, multistage fermentation optimization, factorial design, and single-factor experiments were employed to identify and refine the key nutritional components and culture conditions influencing nematicidal activity. The results showed that the corrected mortality of pine wood nematodes reached 89.75% with the bacterial fermentation suspension (BFS) and 84.50% with the bacterial fermentation filtrate (BFF), with BFS exhibiting significantly higher activity. Ultrastructural analysis revealed that Pt-RP9 induced cuticle shrinkage and rupture, accompanied by lipid droplet fragmentation (particle size reduced from 9.78 µm to 1.24 µm). Factorial analysis identified yeast extract concentration, sodium chloride concentration, and fermentation temperature as the major influencing factors. The optimized fermentation conditions were determined as yeast extract 15 g·L⁻¹, peptone 10 g·L⁻¹, sodium chloride 7.5 g·L⁻¹, fermentation temperature 24 ℃, liquid volume 30 mL, and inoculation ratio 1:30. Under these conditions, the nematicidal activity of Pt-RP9 increased to 94.38–97.36%. In conclusion, this study clarified the nematicidal characteristics and mechanism of Pt-RP9 and established an efficient fermentation process, providing a scientific basis for the development of Pt-RP9-based biocontrol agents and their application in the eco-friendly management of pine wilt disease.