Periphyton is frequently used in the evaluation of the ecological status of aquatic ecosystems using diatoms as a proxy. However, periphyton has a particularity, the production of extracellular polymeric substances (EPS), which might play a protective role against exposure to harmful environmental contaminants. Effluents originating in wastewater treatment plants (WWTPs) constitute some of the most complex mixtures of contaminants, to which aquatic ecosystems are frequently exposed, often containing tens to hundreds of different chemicals. In such challenging scenarios, a putative protective role of EPS may obscure the bioindicator value of diatoms. To address this problem, we sampled periphyton upstream and downstream of the effluent outfall from three different WWTPs, quantifying EPS production and simultaneously evaluating general stress responses in the community (protein and sugar content, photosynthetic pigments, antioxidant enzyme activity and oxidative damage). By combining these endpoints with a characterization of the sediments of the riverine systems receiving the effluents made in a previous study (metals, polycyclic aromatic hydrocarbons, pharmaceuticals and personal care products), we aimed to elucidate whether effluent contaminants trigger negative effects, which may be mitigated by EPS layers protecting the communities. Our results indicated that under a comparatively milder contamination burden, EPS production is enhanced in samples collected downstream of the effluent outfall; under a higher contamination burden, EPS production is hampered. Stress-coping mechanisms were activated by environmental contaminants, including the antioxidant defense, particularly through catalase and superoxide dismutase activity. The findings support the generally assumed protective effect of EPS, but also suggest that EPS production depends on the contamination burden and that protective effects should be in place under specific scenarios of, for example, relatively low contamination levels. Overall, the integrative approach used in this study contributes to a better understanding of the complex interplay of interactions between effluent-driven contamination and thriving periphytic communities inhabiting recipient waterways, including evolved protection mechanisms.