Fragmentation and habitat loss driven by agricultural expansion have significantly affected bird ecology. Additionally, the growing use of agrochemicals impacts ecosystems and avian communities. Tools such as isotopic analysis provide reliable proxies for assessing organisms’ habitat use and diet, and can also reflect environmental alterations, such as increased input of agrochemicals and shifts in baseline resources. By employing stable isotope analysis of carbon (δ13C) and nitrogen (δ15N) on bird feathers over an extensive timescale, we investigated whether human impacts have changed their habitat use, diet, and feather development in two key regions of the Brazilian Atlantic Forest: the Pernambuco and Serra do Mar areas of endemism. We analyzed feathers from 1,778 museum specimens, categorized by region, trophic guild, and temporal groups (historical and modern). Furthermore, we examined fault bars in the wing and tail feathers of 856 specimens to assess changes in feather development. In Serra do Mar, comparisons showed a significant decline in δ15N over time, while δ13C remained stable. These findings suggest that, in addition to habitat loss, agrochemical runoff may indirectly affect bird communities in this region, known for its high agricultural activity. In contrast, isotopic changes in Pernambuco were significant for only a few species, suggesting that habitat use and diet shifts in this region are primarily driven by fragmentation and forest loss. Moreover, we noted a decline in feather quality over time, underscoring the negative effects of human stressors on feather development across the Atlantic Forest birds.