The hydrological effects of forest cover loss are difficult to discern in the case of large‐scale basins with gradual changes and difficult to isolate when climate variability is also present. In the present study, we evaluated the effects of climate variability and human activity on the annual streamflow in a basin in the Amazon arc of deforestation. We statistically analyzed the components of the annual water balance and monthly streamflow and used the currently used Tomer‐Schilling, elasticity, and decomposition of Budyko‐type curve methods to separate climate‐induced changes and anthropogenic effects. Annual series of the monthly maximum and minimum streamflow, total streamflow, and total reference evapotranspiration presented statistically significant increasing trends. No significant trend was observed for precipitation. The greatest change in the average annual runoff coefficient was observed between the first (1973–1984) and second (1985–1994) analyzed periods. Even with the continuous reduction in the forested area, the third (1994–2004) and fourth analyzed periods (2003–2016) showed only relatively small changes, most likely due to the intensity of slash‐and‐burn activities and vegetation regrowth. The methods showed that deforestation was the primary cause of the streamflow changes, but with different intensities, and a small recuperation was observed in the last analyzed period. On average, the annual water yield would increase between 26% and 58% after the first time interval without the opposite effect of climate variability, which must be considered in basin management. Future research should focus on analyzing the water storage and the dependence of the precipitation‐runoff relationship from the climate.