Resumo. Abstract: Understanding the relationship between environmental quality of streams and biological integrity of fish assemblages is critical to successful ecosystem management. Aim We adapted the Index of Biotic Integrity (IBI) using ecological data of the fish assemblages that occur in headwater streams from the Atlantic Forest in southern São Paulo State. Methods We sampled the ichthyofauna and collected environmental data in 27 streams stretches during the dry season of 2010. The fish species were categorized into trophic group, position in the water column and preference for rapid meso-habitats. Candidate metrics were screened for range, responsiveness and redundancy. Results Of the 17 metrics tested, four metrics were included in the IBI. They belonged to attributes species diversity: percentage of individuals as Loricariidae family; habitat use: percentage of individuals as benthic riffles; and trophic function: percentage of individuals as omnivores and percentage of individuals as herbivores/detritivores. Eight streams (30%) were classified as excellent or good and fourteen (50%) as poor or very poor. Conclusions On a regional scale, many aspects of biological integrity were altered but there are streams that can be used as biological reference.