Abstract In urban areas, aquatic ecosystems and their communities are exposed to numerous stressors of various natures (chemical and physical), which impacts are often poorly documented. In epidemic context such as the COVID 19 pandemic, the use of biocides such as the dodecylbenzyldimethylammonium chloride (BAC 12) increased, resulting in an expectable increase in their concentrations in urban aquatic ecosystems. This compound is known to be toxic to most aquatic organisms. Besides, artificial light at night (ALAN) is increasing globally, especially in urban areas. ALAN may have a negative impact on photosynthetic cycles of periphytic biofilms, which in turn may result in changes in their metabolic functioning. Moreover, studies suggest that exposure to artificial light could increase the biocidal effect of BAC 12 on biofilms. The present study investigates the individual and combined effects of BAC 12 and/or ALAN on the functioning and structure of photosynthetic biofilms. We exposed biofilms to a nominal concentration of 30 mg.L-1 of BAC 12 and/or ALAN for 10 days. BAC 12 had a negative impact on biofilm functioning by decreasing the amount and the quality of photosynthetic pigments, resulting in a >90% decrease in photosynthetic efficiency after 2 days of exposure. We also noted a strong decrease in glycolipids that resulted in a shift in lipid profiles. We found no significant effect of ALAN on the endpoints assessed and no interaction between the two stress factors. Graphical abstract