Ecotoxicological tools have proved to be sensitive and appropriate for the evaluation of the effectiveness of treatments used in wastewater treatment plants (WWTP). The objective of this study was to assess the applicability of bioassays and biomarkers to evaluate the efficiency of different treatments throughout WWTP samples[A—raw influent, B—preliminary effluent, C—final effluent, and D—receiving stream], seasonally over 1 year, through a multispecies approach: i) bacterial cell viability [Escherichia coli, Rhodopirellulla rubra, Arthrobacter sp., and Pseudomonas putida]; ii) microalgae Raphidocelis subcapitata and the macrophyte Lemna minor growth inhibition; and iii) microcrustacean Daphnia magna acute and feeding rate assays. Total chlorophyll, malondialdehyde, and proline levels were evaluated in L. minor, and catalase, glutathione-S-transferase activities, and thiobarbituric acid reactive substances levels were quantified in D. magna, after exposure to wastewater samples. Overall, the tested species showed different sensitivities, P. putida = Arthrobacter sp. = R. rubra < R. subcapitata < E. coli = D. magna = L. minor, to the collected samples. The results obtained in D. magna and L. minor assays demonstrated that these organisms can be used in programs for monitoring and environmental assessment of wastewater effluents. The present study demonstrates the usefulness of ecotoxicological tools, with multispecies and different endpoints, to assess the effectiveness of WWTPs. Moreover, it is important to ensure that WWTP implements a monitoring program to minimize the discharge of effluents that compromise the environment in order to guarantee the good ecological quality of the environmental ecosystems.