Many-year data (1992–2010) of bioassay studies were used to identify the dynamics of surface water toxicity in water bodies of the Lower Don in Rostov-on-Don City. Segments of the Lower Don, the Temernik R. mouth, and Lake Goluboe were studied taking into account the anthropogenic load typical of them. The toxicity was evaluated by bioassay tests with four test-objects with different trophic levels and systematic positions. The toxic pollution of surface waters of the Don R. basin within a megalopolis still persists in the XXI century. However, water toxicity is heterogeneous. Thus, trends toward a considerable decrease or increase in water toxicity were recorded in individual parts of the Lower Don, while the situations remained stable in other parts. Bioassay yields an adequate estimate of water toxicity.