The composition of lipids in algae are significantly influenced by environmental factors, including light intensity. Exposure to organic and inorganic contaminants can also disrupt the synthesis of fatty acids, changing the lipid composition of microalgae in periphytic communities. In this study, we looked at how a biocide such as dodecylbenzyldimethylammonium chloride (BAC 12) and two photoperiod durations can affect a biofilm's polar lipidome in a microcosm experiment. The heterotrophic compartment appeared to be raised by exposure to BAC 12 at the expense of phototrophic organisms. Additionally, the overall decline in polyinsaturated fatty acids indicated that the biofilm's phototrophic organisms were all severely impacted. However, it may be difficult to differentiate the effects of contamination from those of light, since there was no observable effect of photoperiods on the conventional fatty acid determination. The molecular species composition of both glycolipids and phospholipids was investigated in additional multivariate analyses. It was suggested that some molecular species may serve as more specific markers of light duration at the biofilm scale. Lastly, we recommend applying a similar lipidomic approach with monospecific cultures of microalgal strains in future research to support these findings, as the methodology used in this study would be applicable to other biofilm-derived microorganisms.