Resumo. Ants have been extensively used as bioindicators, however ants from different nest stratum and habitat affinity groups could distinctly respond to a same ecological process and environmental impact. In this study, we evaluated if nest stratum and habitat affinity matter in the response of ant assemblages to forest-pasture shifting. We tested the response of number of species in entire ant assemblages (soil surface and subterranean) and in each ant fauna stratum (only soil surface and only subterranean). In both cases, we also tested the response of number of ant species of each habitat affinity groups (forest specialist, open-habitat specialist and generalist). Ants were sampled in three plots for each habitat type in Southwestern Brazilian Amazon. We sampled 124 ant species. Only for soil surface ant assemblages, the number of species was different between the two habitats types, among habitat affinity ant groups and their number of species also changed with habitat shifting. Therefore, we corroborate the inclusion of samplings in different nest stratum and recommend the classification of ants according to their habitat affinity in monitoring programs that use ants as bioindicator. However, efforts must be done to improve the information availability on habitat affinity of ant species.