Fluctuating resources and conditions can regulate the community structure of ants, affecting their activities and interactions. This has important implications for the selection of sampling periods during bioindication studies. The aim of this study was to evaluate if the chosen sampling period influences the response of ant assemblage diversity and ecological function to mining impacts and rehabilitation age after mining. We used ant assemblage parameters, including arboreal, epigaeic and hypogaeic ant species richness and composition, seed-removing ant species richness and composition, and seed removal rate in areas impacted by mining and with different rehabilitation ages. We showed that in most cases the response is the same, regardless of sampling period. However, we suggest that ant sampling is best undertaken in the rainy period, when ant species richness reaches higher values and the assemblage composition presents a well-marked difference among the areas. We also indicate that the epigaeic ant assemblage is sufficient for evaluating mining impacts, but arboreal strata should also be sampled in rehabilitation scenarios.