The fate and effects of cytostatic (anticancer or antineoplastic) pharmaceuticals in the environment are largely unknown, but they can contaminate wastewater treatment effluents and consequently aquatic ecosystems. In this paper, we have focused on five cytostatic compounds used in high amounts (cyclophosphamide, cisplatin, 5-fluorouracil, doxorubicin, and etoposide), and we have investigated their ecotoxicity in bacterial Pseudomonas putida growth-inhibition test, algal Pseudokirchneriella subcapitata growth-inhibition test, and Dapnia magna acute immobilization test. Genotoxicity also was assessed with Escherichia coli SOS-chromotest (with and without metabolic activation) and the GreenScreen Assay using yeast S. cerevisiae. All tested compounds showed significant effects in most of the assays with lowest-observed-effect concentrations and concentrations causing 50% effects (EC50s) values ranging within microg/L to mg/L. The most toxic compound was 5-fluorouracil in the assays with P. putida (EC50 = 0.027 mg/L) and P. subcapitata (EC50 = 0.11 mg/L), although cisplatin and doxorubicin were the most toxic to D. magna (EC50 = 0.64 and 2.0 mg/L, respectively). These two chemicals were also the most genotoxic in the SOS-chromotest (minimum genotoxic concentrations [MGC] = 0.07-0.2 mg/L), and 5-fluorouracil was the most genotoxic in the eukaryotic yeast assay (MGC = 0.02 mg/L). Our investigation seems to indicate generally lower risks of acute effects at concentrations expected in the environment. However, some effective concentrations were relatively low and chronic toxicity of cytostatics (and/or their transformation products), as well as specific sources of human pharmaceuticals such as hospital effluents, require research attention.