Quantifying and understanding the main drivers of biodiversity responses to human disturbances at multiple scales is key to foster effective conservation plans and management systems. Here we report on a detailed regional assessment of the response of ant communities to land-use change and forest disturbance in the Brazilian Amazon. We aimed to explore the effects of land-use intensification at both site and landscape scales, examining variation in ant species richness and composition, and asking which set of environmental variables best predict observed patterns of diversity. We sampled 192 sites distributed across 18 landscapes (each 50 km2) in Paragominas, eastern Brazilian Amazon, covering ca. 20,000 km2. We sampled from undisturbed primary forest through varyingly disturbed primary forests, secondary forests, pastures and mechanised agriculture, following a gradient of decreasing total aboveground biomass. Irrespective of forest disturbance class, ant species richness was almost twice as high in forests when compared to production areas. In contrast, ant species composition showed continuous variation from primary forest to intensive agriculture, following a gradient of aboveground biomass. Ant species richness at all spatial scales increased with primary forest cover in the surrounding landscapes. We highlight the limited value of species richness as an indicator of changes in habitat quality, reinforcing calls to consider species composition in assessments of forest disturbance. Taken together, our results reveal the unique biodiversity value of undisturbed primary forests, but also show that disturbed primary forests and secondary forests have high conservation value, and thus play an important role in regional conservation planning.