Ecotoxicological experiments were performed in laboratory-scale microcosms to investigate community-level responses of river phototrophic biofilms from different environments to herbicide exposure. Biofilms were initially cultivated on artificial substrates placed in situ for 4 weeks at two sites, site M, located in an agricultural watershed basin and site S, located in a forested watershed basin. The biofilms were subsequently transferred to microcosms and, after an acclimatization phase of 7 days, were exposed to alachlor at 10 and 30 µg L-1 for 23 days. Alachlor effects were assessed by a combination of structural parameters, including biomass (ash free dry mass and chlorophyll a), molecular fingerprinting of the bacterial community (polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and diatom species composition. Alachlor impacted the chlorophyll a and ash-free dry mass levels of phototrophic biofilms previously cultivated at site S. The structural responses of bacterial and diatom communities and diatom were difficult to distinguish from changes linked to the microcosm incubation period. Phototrophic biofilms from site S exposed at 30 µg L-1 alachlor were characterised by an increase of Achnanthidium minutissimum (K-z.) Czarnecki abundance, as well as a higher proportion of abnormal frustules. Thus, phototrophic biofilms with different histories, exhibited different responses to alachlor exposure demonstrating the importance of growth environment. These observations also confirm the problem of distinguishing changes induced by the stress of pesticide toxicity from temporal evolution of the community in the microcosm.