Quantifying the relative contribution of climate change and anthropogenic activities to runoff alterations are essential for the sustainable management of water resources in Central Asian countries. In the Kofarnihon River Basin (KRB) in Central Asia, both changing climate conditions and anthropogenic activities are known to have caused changes to the hydrological cycle. Therefore, quantifying the net influence of anthropogenic contribution to the runoff changes is a challenge. This study applied the original and modified Mann–Kendall trend test, including the Sen’s slope test, Pettitt’s test, double cumulative curve, and elasticity methods. These methods were applied to determine the historical trends, magnitude changes and change points of the temperature, precipitation, potential evapotranspiration, and runoff from 1950 to 2016. In addition, the contributions of climate change and anthropogenic activities to runoff changes in the KRB were evaluated. The trend analysis showed a significant increasing trend in annual temperature and potential evapotranspiration, while the annual precipitation trend showed an insignificant decreasing trend during the 1950–2016 time period. The change point in runoff occurred in 1986 in the upstream region and 1991 in the downstream region. Further, the time series (1950–2016) is separated into the prior impacted period (1950–1986 and 1950–1991) and post impacted period (1987–2016 and 1992–2016) for the upstream and downstream regions, respectively. During the post impacted period, climate change and anthropogenic activities contributed to 87.96% and 12.04% in the upstream region and 7.53% and 92.47% in the downstream region of the KRB. The results showed that in runoff changes, the anthropogenic activities played a dominant role in the downstream (97.78%) and the climate change impacts played a dominant factor in the upstream region (87.96%). In the land-use type changes, the dominant role was played by construction land, which showed that the area from 248.63 km2 in 1990 increased to 685.45 km2 (175.69%) in 2015. These findings suggest that it is essential to adopt effective steps for the sustainable development of the ecological, hydrological, and social order in the KRB in Central Asia.