Metals are naturally present in freshwater ecosystems but anthropogenic activities like mining operations represent a long-standing concern. Metals released into aquatic environments may affect microbial communities such as periphytic biofilm, which plays a key role as a primary producer in stream ecosystems. Using two 28-day microcosm studies involving two different photoperiods (light/dark cycle of 16/8 vs 8/16), the present study assessed the effects of four increasing nickel (Ni) concentrations (0-6 mu M) on two natural biofilm communities collected at different seasons (summer and winter). The two communities were characterized by different structural profiles and showed significant differences in Ni accumulated content for each treatment. For instance, the biofilm metal content was four times higher in the case of summer biofilm at the highest Ni treatment and after 28 days of exposure. Biomarkers examined targeted both heterotrophic and autotrophic organisms. For heterotrophs, the 8-glucosidase and 8-glucosaminidase showed no marked effects of Ni exposure and were globally similar between the two communities suggesting low toxicity. However, the photosynthetic yield confirmed the toxicity of Ni on autotrophs with maximum inhibition of 81 +/- 7% and 60 +/- 1% respectively for the summer and winter biofilms. Furthermore, biofilms previously exposed to the highest long-term Ni con-centration ([Ni2+] = 6 mu M) revealed no acute effects in subsequent toxicity based on the PSII yield, suggesting a tolerance acquisition by the phototrophic community. Taken together, the results suggest that the biofilm response to Ni exposure was dependent of the function considered and that descriptors such as biofilm metal content could be seasonally dependent, information of great importance in a context of biomonitoring.