Freshwater ecosystems are subjected to a broad range of anthropogenic threats. Understanding the responses of aquatic biota to these threats and identifying potential mitigating actions presents a major challenge for ecologists and natural resource managers. Here, we investigated the responses of fish communities to land use change at different scales. We assessed patterns of food web structure of fish across a gradient of human impacts in the Atlantic Forest ecoregion of southeast Brazil. We analysed stomach contents of fish and evaluated the response of five food web metrics to variation in land use change. We also calculated four metrics for intra-guild comparisons in the omnivore, insectivore, and algivore–insectivore guilds. The proportional abundance of omnivores increased as temperature increased and canopy cover decreased. Food web specialisation decreased in deforested streams with higher nutrient input, suggesting higher resource sharing by those fish communities. Fish in the algivore–insectivore guild consumed fewer types of macroinvertebrates in streams with low canopy cover. The opposite pattern was observed within the insectivore guild with individuals consuming a larger variety of prey in impacted streams. Deforestation, and increases in water temperature and nutrient input, influenced inter-species and intra-guild trophic interactions in fish communities in these Atlantic Forest streams, even though the richness of fish community and composition of intra-guild species remained unchanged. Some food web metrics were sensitive to the direct and indirect effects of disturbance on these fish communities. Our study has shown that assessments of trophic interactions can detect the effects of land-use disturbance on fish communities that may not be evident from simple measures of species richness and composition. This approach provides direct insights into the functional integrity of fish communities and should be considered for monitoring the success of programmes aimed to protect and, where necessary, restore freshwater ecosystems.