Water scarcity is a key challenge to global development. In Brazil, the Sao Francisco River Basin (SFB) has experienced water scarcity problems because of decreasing streamflow and increasing demands from multiple sectors. However, the drivers of decreased streamflow, particularly the potential role of the surface-groundwater interaction, have not yet been investigated. Here, we assess long-term trends in the streamflow and baseflow of the SFB during 1980–2015 and constrain the most likely drivers of observed decreases through a trend analysis of precipitation (P), evapotranspiration (ET), and terrestrial water storage change (TWS). We found that, on average, over 86% of the observed decrease in streamflow can be attributed to a significant decreasing baseflow trend along the SFR, with a spatial agreement between the decreased baseflow, increased ET, and irrigated agricultural land in the Middle SFB. We also noted a decreasing trend in TWS across the SFB exceeding –20 mm year−1. Overall, our findings indicate that decreasing groundwater contributions (i.e., baseflow) are providing the observed reduction in the total SFR flow. A lack of significant P trends and the strong TWS depletion indicate that a P variability only has likely not caused the observed baseflow reduction, in mainly the Middle and Sub-middle SFB. Therefore, groundwater and surface withdrawals may likely be a driver of baseflow reduction in some regions of the SFB.