The river basin of Siversky Donets is of great scientific interest since this river runs through a territory with heavy industry (in particular, coal mining, chemical processing and metal industries). Within the basin, rivers of different sizes were explored (small, medium, large and extra-large) that flow through siliceous and calcareous rocks on the same elevation (lowland—below 200 m a.s.l.). Phytoplankton, as one of the Biological Quality Element, was used to perform the assessment of ecological status of the water bodies within the Siversky Donets river basin in 2019. The state monitoring program based on the updated approaches has been implemented in the river basin for the first time. The composition of phytoplankton species in the basin comprised 167 species (168 intraspecies taxa), mainly Bacillariophyta (63%) and Chlorophyta (22%) with the presence of other species (Cyanobacteria, Charophyta, Chrysophyta, Dinophyta and Euglenophyta). High species diversity and divisions amount are a distinctive property of the smaller rivers, while the bigger rivers show lower number of divisions. The “bloom” events, which are important ecological factors, were not detected in the Siversky Donets river basin. Algal species composition in plankton samples of the basin was identified and series of ecological parameters, such as habitat preferences, temperature, pH, salinity, oxygenation and organic water pollution according to Watanabe and Sládeček’s index of saprobity (S) trophic state and nitrogen uptake metabolism were analyzed. The ecological conclusions were also verified by a canonical correspondence analysis (CCA). The significance of the Canonical Correspondence Analysis (CCA) results was estimated of by a Monte-Carlo permutation test. The high concentrations of inorganic phosphorus compounds (permanganate index (CODMn)) and nitrite ions favored the diversity of Chlorophyta and Cyanobacteria diversity correlated with the levels of bicarbonate and CODMn. High diversity of diatoms was facilitated by the total amount of dissolved solids and chemical oxygen demand (COD). It was found that low water quality could be associated with conditions leading to predominant growth of the mentioned groups of algae. According to the analysis, the highest water quality was characterized by balanced phytoplankton composition and optimal values of the environmental variables. The sites with reference conditions are proposed for future monitoring.