While legal regulations require treated wastewater to be tested based only on its physicochemical parameters, surface water assessment also has to include biological indicators. However, neither approach provides a complete picture of water quality due to lack of ecotoxicological information. Therefore, the aim of the study was to perform an ecotoxicological evaluation of treated wastewater and river water in the catchment scale using a battery of biotests. In the period between June 2017 and July 2018, six sets of treated wastewater samples were taken from 17 municipal wastewater treatment plants (WWTPs) of different sizes (small: < 2,000 people equivalent - p.e.; medium size: 2,000–9,999 p.e.; large: 15,000–99,999 p.e.), as well as river water samples from seven sites along the 342 km Pilica River in central Poland. Physical and chemical analysis were performed of total nitrogen (TN), total phosphorus (TP), biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total suspended solids (TSS), as well as organic suspended matter (OSM), ion content (fluorides, nitrates, ammonium, potassium and sulphate), and pH, conductivity and redox potential (RP). Ecotoxicity assessment was performed using a battery of biotests comprising the producer Pseudokirchneriella subcapitata (chronic toxicity biotest), consumer Thamnocephalus platyurus (acute toxicity biotest) and decomposer Tetrahymena thermophila (chronic toxicity biotest). It was found that in small WWTPs, the recommended levels of TN, TP, BOD, COD and TSS were often exceeded. Also, the highest mean toxicity hazard in the ecotoxicological biotests was observed for samples from small WWTPs. No clear dependence was found between any of the studied physicochemical parameters of the water and the ecotoxicity of samples: however, a correlation was observed between growth inhibition of P. subcapitata and TN and NH4+ concentration (r = 0.43 and r = 0.48, respectively). Also, the mortality of T. platyurus was correlated with NH4+(r = 0.72), TN (r = 0.64), BOD (r = 0.52), TSS (r = 0.44) and OSM (r = 0.46). The most sensitive organism in the applied battery of biotests was the alga P. subcapitata (producer), with a 90% toxic response for WWTP samples and 100% for river samples. T. platyurus (consumer) demonstrated a 56% toxic response for WWTP samples and no toxicity (0%) for river samples. In turn, T. thermophila demonstrated a 25% toxic response for WWTP samples and 33% for river samples. The addition of an ecotoxicological evaluation (battery of biotests) to the physicochemical monitoring of treated wastewater and river water delivered new information about the biological impact on organisms related to their different sensitivity. Additionally, the small WWTPs turned out to be the highest source of hazard.