Abstract Background Several biomarkers developed from freshwater sentinel species are used to complement chemical assessment of freshwater ecosystems and improve the evaluation of their contamination levels. While active biomonitoring strategies allow to compare biological data in the field, the lack of assessment criteria to interpret biomarkers modulations restricts their application in freshwater biomonitoring surveys. For this purpose, the determination of threshold values emerges as a relevant approach to support the biomarker-based diagnosis within biomonitoring surveys. Based on the distribution of contamination baselines, a statistical approach was proposed to define threshold values of bioaccumulated concentrations in the sentinel species Gammarus fossarum . Although this methodology is limited to chemical databases, this approach could be adapted for defining threshold values at the biological level. This study aims to demonstrate the possibility of defining threshold values for different classes of biomarkers monitored in two freshwater sentinel species ( Dreissena polymorpha , G. fossarum ) and evidence the added value of such an approach in biomarker-based diagnosis. Results For this purpose, the study benefited from exceptional environmental biomarker datasets acquired from mussels and gammarids caged in hundred environmental sites, representative of the diversity of French water bodies. Thanks to these environmental datasets, inhibition and/or induction thresholds could be defined for 8 biochemical, cellular, and molecular biomarkers of D. polymorpha and 25 peptide biomarkers of G. fossarum, not influenced by confounding factors. Threshold values were then applied to another biomarker dataset acquired from mussels and gammarids caged upstream and downstream four wastewater treatment plants (WWTPs). Several biomarkers were modulated both downstream and upstream of WWTPs, indicating a potential contamination not related to WWTPs. In contrast, some biomarkers were impacted exclusively downstream of the WWTPs, highlighting an impact of WWTP discharges on the aquatic fauna. Conclusions This study demonstrates the possibility to define threshold values for a wide range of biomarkers (cellular, biochemical and molecular) monitored on different sentinel species. The definition of threshold values constitutes an attractive solution to support the application of biomarkers in active biomonitoring surveys and improve the biomarker-based diagnosis without the need of “reference sites”.