α-terthienyl is an allelochemical derived from the roots of marigold (Tagetes spp.), which is used to suppress plant parasitic nematodes. We investigated the nematicidal activity of α-terthienyl against the model organism Caenorhabditis elegans and the root-knot nematode, Meloidogyne incognita. As reported previously, α-terthienyl action was much higher after photoactivation, but was still effective against C. elegans dauer larvae and M. incognita second stage juveniles even without photoactivation. Expression induction of two major enzymes, glutathione S-transferase (GST) and superoxide dismutase (SOD), was restricted in C. elegans hypodermis following treatment with α-terthienyl. The susceptibility of nematodes to α-terthienyl changed when the expression of GST and SOD was induced or suppressed. From these results, under dark conditions (without photoactivation), α-terthienyl is an oxidative stress-inducing chemical that effectively penetrates the nematode hypodermis and exerts nematicidal activity, suggesting high potential for its use as a practicable nematode control agent in agriculture.