Grammicin, a polyketide metabolite produced by the endolichenic fungus Xylaria grammica KCTC 13121BP, shows strong nematicidal activity against Meloidogyne incognita. This study was performed to elucidate the grammicin biosynthesis pathway of X. grammica KCTC 13121BP and to examine the nematicidal activity of the biosynthesis intermediates and derivatives against M. incognita. Two grammicin biosynthesis intermediates were isolated from a T-DNA insertion transformant (strain TR-74) of X. grammica KCTC 13121BP and identified as 2-(hydroxymethyl)cyclohexa-2,5-diene-1,4-dione (compound 1) and 2,5-dihydroxybenzaldehyde (compound 2), which were also reported to be intermediates in the biosynthesis pathway of patulin, an isomer of grammicin. This indicates that the grammicin biosynthesis pathway overlaps almost with that of patulin, except for the last few steps. Among 13 grammicin biosynthesis intermediates and their derivatives (except grammicin), toluquinol caused the highest M. incognita J2 mortality, with an LC50/72 h value of 11.13 µg/mL, which is similar to grammicin with an LC50/72 h value of 15.95 µg/mL. In tomato pot experiments, the wettable powder type formulations (WP) of toluquinol (17.78 µg/mL) and grammicin (17.78 µg/mL) also effectively reduced gall formation on the roots of tomato plants with control values of 72.22% and 77.76%, respectively, which are much higher than abamectin (16.67%), but lower than fosthiazate (100%). The results suggest that toluquinol can be used directly as a biochemical nematicide or as a lead molecule for the development of new synthetic nematicides for the control of root-knot nematode diseases.