Study region: Madeira River basin, southwestern Amazonia Study focus: This study investigates spatial and temporal changes in precipitation, evaporation, and streamflow, and their relationship with deforestation in the Madeira River basin, the largest Amazonian sub-basin. We applied Mann-Kendall trend analysis, change-point detection, and correlation analysis across multiple spatial scales, using satellite, reanalysis, and observed data from 1981 to 2015. These methods enabled us to detect long-term trends, identify shifts, and quantify the relationships between forest loss and hydrological changes New hydrological insights for the region: The basin experienced an average deforestation rate of 2810 km² per year from 2001 to 2020, predominantly in the Brazilian portion. Between 1981 and 2016, we observed statistically significant negative trends in precipitation, evaporation, and streamflow, especially in the most deforested areas during the wet season. Correlation analysis (2001–2015) showed a statistically significant and positive relationship between forest area and evaporation in wet months (r = 0.73, p < 0.1) and a negative correlation between forest area and streamflow during the same season (r = –0.6, p < 0.1). These findings highlight the critical role of forests in modulating hydrological processes, supporting the hypothesis that deforestation may reduce evaporation, alter moisture recycling, and slow the water cycle. While our results are robust, we acknowledge that factors such as climate variability and land management practices may also influence hydrological changes and should be considered in future research.