Photosynthetic performance of algal-bacterial biofilms from an Italian wastewater treatment plant was studied in a flow-lane photobioreactor at different irradiances, temperatures, and flow regime to evaluate the effects of these environmental parameters on biofilms' functioning, in view of application of these communities in wastewater biological treatment. Pulse amplitude modulated fluorescence was used to estimate the effective quantum yield of PSII (Delta F/F-m') of the light-acclimated biofilms and to perform rapid light curves (RLCs) for the determination of the photosynthetic parameters (rel.ETRmax, alpha, I-k). Chl a, ash free dry weight (AFDW), and dry weight (DW) were measured to assess phototrophic and whole biofilm biomass development over time. From the analysis of photosynthetic parameter variation with light intensity, temperature and flow rate, it was possible to identify the set of experimental values favoring biofilm photosynthetic activity. Biomass increased over time, especially at the highest irradiances, where substrata were fastly colonized and mature biofilms developed at all temperatures and flow conditions tested.