The complex interplay between species along environmental gradients ultimately shapes their distributions and additional community interactions. Ant-mediated seed dispersal fails in the wettest habitat of deciduous forest in eastern North America, and we examine whether this pattern corresponds with colony distributions for seed-dispersing ants and associated heterogeneity in abiotic and biotic variables. Specifically, we used spatial variation in soil moisture, temperature and diffuse light along natural habitat gradients and experimentally manipulated soil moisture gradients to examine ant habitat selection. We also examined niche segregation between effective (Aphaenogaster spp.) and ineffective (Lasius alienus Foerster) seed-dispersing ants across these environmental gradients. Whereas most research links ant foraging and nesting with temperature gradients, we find niche segregation between Aphaenogaster spp. and L. alienus by soil moisture along naturally occurring gradients and in experimentally irrigated upland habitat. The failure of Aphaenogaster spp. to occupy the wettest habitats, where L. alienus is present, is consistent with observed seed dispersal failure in these habitats. These results indicate that environmental heterogeneity drives niche segregation between effective (Aphaenogaster spp.) and ineffective (L. alienus) seed dispersers so each occupies distinct habitat. Most forest understory plants rely on ants for seed dispersal. Our research implies that climate-mediated interactions between effective and ineffective seed dispersing ant species may structure the microhabitat distributions for woodland herbs.