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The essential oil (EO) was extracted from aerial parts with insecticidal and fungicidal activity. Herein, the hydro-distilled essential oils of Seseli mairei H. Wolff roots were determined by GC-MS. A total of 37 components were identified, (E)-beta-caryophyllene (10.49%), β-geranylgeranyl (6.64%), (E)-2-decenal (6.17%) and germacrene-D (4.28%). The essential oil of Seseli mairei H. Wolff had nematicidal toxicity against Bursaphelenchus xylophilus with a LC50 value of 53.45 μg/mL. The subsequent bioassay-guided investigation led to the isolation of three active constituents: falcarinol, (E)-2-decenal, and octanoic acid. The falcarinol demonstrated the strongest toxicity against B. Xylophilus (LC50 = 8.52 μg/mL). The octanoic acid and (E)-2-decenal also exhibited moderate toxicity against B. xylophilus (LC50 = 65.56 and 176.34 μg/mL, respectively). The LC50 of falcarinol for the toxicity of B. xylophilus was 7.7 and 21 times than that of octanoic acid and (E)-2-decenal, respectively. Our findings demonstrate that the essential oil from Seseli mairei H. Wolff roots and their isolates may be developed as a promising natural nematicide.