The natural vulnerability characterization provides fundamental support for identification of areas more susceptible from the hydrology point of view, assuring the maintenance of water resource features. In this context, this study aimed to characterize the natural vulnerability of water resources based on long-term streamflows, base flow and aquifers’ susceptibility to contamination, for Formoso River basin, located in southwestern Tocantins. For that, subdivision by level 5 Ottobasins and discharge and precipitation data sets, both available from the “Brazilian National Water Agency”, and the geological map developed by “Tocantins State Bureau of Planning” (SEPLAN) were used. The final natural vulnerability of water resources presented degrees varying from low to very high. Areas with lower vulnerability were observed in the basin headwaters, mainly due to the greatest both long-term (SYqlt) and 90% of the exceedance (SY90%) specific discharges. “High” vulnerability were identified in most of the middle basin course while “Very High” vulnerability in the lower basin course given by lower SYqlt and SY90% and by the occurrence of the alluvial aquifers which present high natural susceptibility to contamination.