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This work focuses on the application of the protocol for the assessment of the environmental footprint of products and organisations, performed accordingly with the prescriptions of the European Commission Recommendation 2013/179/EU. Although scientific and technical literature already reports some example cases carried out in several industrial fields, this methodology has not been run yet for the evaluation of the environmental performances of a wastewater treatment plant. The research has been carried out on a real scale plant, by acquiring, in parallel, operation data, chemical, physical and biological parameters throughout a 2-week campaign. Effluent toxicity towards the aquatic ecosystem was measured, by adopting a multi-tiered approach, the test organisms being crustaceans, bacteria and algae, respectively. The protocol for the evaluation of the environmental footprint was followed by considering as input data either the results of the chemical, physical and biological analyses or those deriving from the bioassays execution. The findings clearly illustrate the invaluable importance of bioassays in the process of evaluation of the environmental impact of any work, together with the need of combining different tests based on specific endpoints and involving organisms playing different trophic roles. [ABSTRACT FROM AUTHOR]