The Serra da Mantiqueira Environmental Protection Area is one of the most important Atlantic Forest biodiversity hotspots in the World, and one of the main water sources for the Brazilian Southeast Region. In this study, we analyze the existence and characteristics of streamflow changes in two watersheds (Sapucaí-Mirim and Sapucaí-Guaçu River watersheds) of the Serra da Mantiqueira Range, and if these changes are due to climate change, alterations of land cover and/or external factors. Mann-Kendall and Pettit tests were used to detect changes in streamflow, rainfall and evapotranspiration data series, at different time scales. The characteristics and magnitude of land cover changes were evaluated from multi-temporal maps, produced from satellite images and the supervised classification technique. The results show an increase in winter rainfall at the two watersheds studied. No changes were detected in the streamflows of the Sapucaí-Mirim River Basin. The summer streamflow of Sapucaí-Guaçu River watershed reduced at a rate of −0.035 m³.s−1. year−1. In addition, there were sudden streamflow changes in the summer (from the year 1967) and autumn (from the year 1988) in Sapucaí-Guaçu watershed. Furthermore, the reference summer evapotranspiration of the Sapucaí-Guaçu watershed showed an increasing trend and an abrupt increase (in 1988), besides a sudden reduction of actual evapotranspiration in 2006. The integrated analysis of these results, together with the changes in land cover and population, allow us to conclude that the reductions in summer and autumn streamflows at the Sapucaí-Guaçu watershed were probably due to the increase in the water withdrawal for public supply, added to the effects of urbanization on the hydrology of the watershed.