Abstract Although the effects of plant secondary metabolites on plant defence have been studied for decades, the exact roles of secondary metabolites in shaping plant-associated microbial and nematode communities remain elusive. We evaluated the effects of benzoxazinoids, a group of secondary metabolites present in several cereals, on root-associated nematodes. We employed 18S rRNA metabarcoding to compare maize root-associated nematode communities in a bx1 knockout maize line impaired in benzoxazinoid synthesis and in its parental wild type. Both genotype and plant age affected the composition of the nematode community in the roots, and the effects of benzoxazinoids on nematode communities were stronger in the roots than in the rhizosphere. Differential abundance analysis and quantitative PCR showed that the root lesion nematode Pratylenchus neglectus was enriched in the bx1 mutant line, while another root lesion nematode, Pratylenchus crenatus, was reduced. Correlation analysis showed that benzoxazinoid concentrations in maize roots mostly correlated negatively with the relative abundance of nematode sequence reads. However, positive correlations between benzoxazinoids and nematode taxa, including several plant-parasitic nematodes, were also identified. Our detailed nematode community analysis suggests differential and selective effects of benzoxazinoids on soil nematodes depending on both the nematode species and the benzoxazinoid compound.