From December 2003 to September 2004, benthic macroinvertebrates (BM), fishes, water and sediment were collected quarterly at six stations in two streams of the upper São Francisco River basin, south-eastern Brazil. We evaluated the ecological conditions, habitat diversity, water quality, composition and structure of BM communities, as well as the food habits of the local fish fauna. By applying a protocol for rapid characterization of ecological conditions and habitat diversity, three of the sampled localities were classified as "pristine" while the others stations were considered "altered". A well oxygenated water with near neutral pH and low electric conductivity (< 0.01 mS/cm) and nutrient concentrations (< 0.08 mg/l total P and < 0.90 mg/l total N) was found for both streams. Sediment analysis revealed the dominance of medium, fine and very fine sand fractions in all sampled areas. The organic matter content in the sediment was higher in the lotic and well preserved area. We found 45 BM taxa, and Chironomidae (68%), Oligochaeta (10%) and Elmidae (8.5%) showed the highest abundances. From the stomach contents analysis of 13 fish species, 26 BM taxa were found, including four that were not collected in the sediment samples, being Chironomidae the dominant group (> 60%). Our results show that human activities such as forest clearing, agriculture and cattle rising have altered the habitat diversity in freshwater ecosystems in a process that affects the aquatic biota and thus the food availability to the fish fauna. The results also highlight the importance of the fish stomach contents analysis as a complementary tool in BM inventories.