Fluvial biofilms are subject to multistress situations in natural ecosystems, such as the co-occurrence of light intensity changes and metal toxicity. However, studies simultaneously addressing both factors are rare. This study evaluated in microcosm conditions the relationship between short-term light intensity changes and Zn toxicity on fluvial biofilms with long-term photoacclimation to different light conditions. Biofilms that had long-term photoacclimation to 25 μmol photons · m−2 · s−1 (low light [LL] biofilms), 100 μmol photons · m−2 · s−1 (medium light [ML] biofilms), and 500 μmol photons · m−2 · s−1 (high light [HL] biofilms) were characterized by different structural (Chlorophyll-a [Chl-a], total biomass-AFDW, EPS, algal groups, and diatom taxonomy) and physiological attributes (ETR-I curves and photosynthetic pigments). HL biofilms showed higher light saturation intensity and a higher production of xanthophylls than LL biofilms. In contrast, LL biofilms had many structural differences; a higher proportion of diatoms and lower AFDW and EPS contents than ML and HL biofilms. A clear effect of light intensity changes on Zn toxicity was also demonstrated. Zn toxicity was enhanced when a sudden increase in light intensity also occurred, mainly with LL biofilms, causing higher inhibition of both the Φ′PSII and the ΦPSII. A decoupling of NPQ from de-epoxidation reaction (DR) processes was also observed, indicating substantial damage to photoprotective mechanisms functioning in biofilms (i.e., xanthophyll cycle of diatoms) due to Zn toxicity. This study highlights the need to take into account environmental stress (e.g., light intensity changes) to better assess the environmental risks of chemicals (e.g., metals)