AbstractAnimals are integrated into the wider ecosystem via their foraging and behavior. The compensation hypothesis predicts that animals target their foraging efforts (i) toward nutrients that are scarce in the environment and (ii) toward nutrients that are not present in the usual diet of species, which varies across trophic levels. Understanding how foraging for resources varies locally, such as across habitat strata, and trophic levels will help to elucidate the links between the local environment and communities to the ecological functions that animals mediate. We examined whether the relative resource use of ants varies consistently along a habitat strata gradient and across trophic levels across Neotropical biomes. We placed 4500 baited tubes, each containing one of five liquid resources (sugar, amino acid, lipid, sodium, and distilled water) in one of three habitat strata (subterranean, epigaeic, and arboreal) across 60 transects in Amazon, Atlantic Forest, Caatinga, Cerrado, Pampa, and Pantanal biomes. We assessed the relative resource use of all ants across the habitat strata and among two different trophic groups across biomes. The relative preference for sugar increased from subterranean to arboreal strata in all biomes, while the relative preference for lipids decreased at this gradient in five biomes. We also found that in general sugar‐consuming ants foraged more for sugar and less for lipids than predatory ants across biomes. Conversely, we found no consistency across biomes in nutrient preference of amino acid and sodium across habitat strata or trophic levels. Overall, our results indicate sugar limitation in the arboreal stratum and lipid limitation on the ground across biomes and that the trophic level of ants strongly determines their foraging efforts—possibly because ants try to fix their dietary nutrient imbalances. Hence, our findings suggest strong local niche partitioning of sugar and lipid use across habitat strata and trophic levels and that other large spatial scale processes influence the local amino acid and sodium dynamics.