An assessment of the Dniester Hydropower Complex (DHPC) impacts on this river streamflow is presented. The study was based on a comparative analysis of Dniester water discharge in periods before (1951–1980) and after (1991–2015) this complex construction, using observation data at hydrological posts located at the entrance to the Dniester reservoir (Zalishchyky) and downstream of its dam (Mohyliv-Podilskyi and Bender). Compared statistics included annual and seasonal trends and averages of water discharge in two periods, and statistical significance of their differences. It was shown that a statistically significant increase of Dniester flow in 1951–1980 was later replaced by its small decrease, explained both by changes in basinwide climate and DHPC functioning manifested in transforming the river flow seasonal distribution. Accumulation of water in the Dniester reservoir has led to a decrease in the annual flow volumes by above 6% directly below its dam and about 9%—in the Lower Dniester. As a result, the role of the Upper Dniester’ catchment, located in the Ukrainian Carpathians, sharply increased; now it provides 80% of the Dniester annual flow compared with 69% before DHPC construction. Another 11% of flow is formed by Dniester’s tributaries in its sub-catchment from Zalishchyky to Mohyliv-Podilskyi and 9%—in its downstream part. Concerning the seasonal streamflow, a challenging reduction is evident in spring due to water accumulation for hydropower needs in DHPC reservoirs, which negatively affects the Low Dniester ecosystems. On the whole, in 1991–2015 the Dniester annual flow decreased from 10.22 to 9.15 km³.