Pine wilt disease, caused by the pine wood nematode (Bursaphelenchus xylophilus), poses a serious threat to forests in Korea. In this study, we investigated the resistance of Pinus densiflora to B. xylophilus following treatment with a fungal elicitor derived from Penicillium chrysogenum through trunk injection and soil drenching. Soil drenching with the fungal elicitor led to increased accumulation of two key defensive compounds: pinosylvin monomethyl ether (PME) and dihydropinosylvin monomethyl ether (DPME) in the needles. In untreated control plants inoculated with B. xylophilus, 67.9% died within five months, whereas only 33.3% of plants pretreated with the elicitor succumbed. Trunk injection of the fungal elicitor also significantly enhanced PME production. The PME concentration in the needles peaked at 155.4 µg/g dry weight (DW) four weeks post-elicitor injection. To assess the impact of elicitor treatment on nematode resistance, we monitored the downward movement of B. xylophilus in plants two months post-injection of fungal elicitor. Nematodes were introduced at the upper internodal segments of branches, and their mobility to the lower segments was measured. In elicitor-treated trees, the movement and population of B. xylophilus were significantly reduced, highlighting the effectiveness of the treatment. These results demonstrate that fungal elicitor treatments, through soil drenching or trunk injection, can enhance the resistance of P. densiflora to B. xylophilus. This eco-friendly approach offers a promising strategy for managing pine wilt disease in susceptible pine species.