The pinewood nematode, Bursaphelenchus xylophilus is a serious forest pathogen in many countries including Japan, China and Korea. To minimize the environmental problems caused by synthetic chemicals broadly utilized in the control of B. xylophilus, we estimated the nematicidal potency of 63 aliphatic compounds against B. xylophilus by measuring their inhibition activity against acetylcholinesterases (ACE, EC 3.1.1.7) of B. xylophilus (BxACEs). In the primary inhibition assay using B. xylophilus crude protein, C6, C9, C10 2E-alkenal C12 alkanoic acid were observed the > 45% BxACE inhibition rate and they were subsequently estimated the inhibition rate against three recombinant BxACEs. Whole compounds showed the high inhibition rate against BxACE-1 and BxACE-2. Interestingly, C6 2E-alkenal and C12 alkanoic acid exhibited the high inhibition rate against BxACE-3 which would be insensitive to ACE inhibitors.