Surface water samples in Ogosta River, Bulgaria and wastewater samples at the inlet and outlet of WWTP–Montana were tested for the traditional parameters and ecotoxicological effect. The river and Dam surface waters comply with category A1 of Directive 75/440/EEC for pH, EC, COD, TSS, NO3–, Cl–, SO42–, B, Ba, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se, V and Zn; with category A2 for BOD5, NH4+–N and Fe; and with category A3 for TNb and As. The average annual concentrations of Al, Cr (III), Cr (VI) and U are lower than the set limits in the Water Framework Directive. Arsenic concentration in all the samples exceeds the maximum allowed concentration, a results from natural processes. All the levels of the studied parameters in the outlet wastewater samples are lower than the limits, set in Directive 91/271/EEC and in the complex permit of the WWTP. The results of the biotest Phytotoxkit F™ show low ecotoxicity of the water samples. Optimization of the sample pretreatment prior to this ecotoxicological test is analyzed and discussed.