The environmental vulnerability diagnosis of a river basin depends on a holistic analysis of its environmental aspects and degradation factors. Based on this diagnosis, the definition of priority areas where interventions for environmental recovery should be carried out is fundamental, since financial and natural resources are limited. In this study, we developed a methodology to assess these fragilities using an Environmental Vulnerability Index (EVI) that combines physical and environmental indicators related to the natural sensitivity of ecosystems and their exposure to anthropogenic factors. The EVI developed was applied to the headwater’s region of the São Francisco River basin, Brazil. The proposed index was based on the AHP multi-criteria analysis and was adapted to include four variables representative of the study area: Land Use Adequacy, Burned Area, Erosion Susceptibility and Quantitative Water Balance. The EVI analysis highlighted that the presence of easily erodible soils, associated with sloping areas and land uses above their capacity, generate the most vulnerable areas in the headwaters of the São Francisco River basin. The results of this study indicate where resources should be applied for environmental preservation in the basin under study, directing the allocation of efforts to areas with lower resilience in maintaining ecosystem services.