Ecotoxicological experiments have been performed in laboratory-scale microcosms to investigate thesensitivity of phototrophic biofilm communities to the alachlor herbicide, in relation to the stages ofphototrophic biofilm maturation (age of the phototrophic biofilms) and physical structure (intact biofilmversus recolonization). The phototrophic biofilms were initially cultivated on artificial supports in aprototype rotating annular bioreactor (RAB) with Taylor-Couette type flow under constant operatingconditions. Biofilms were collected after 1.6 and 4.4 weeks of culture providing biofilms with differentmaturation levels, and then exposed to nominal initial alachlor concentration of 10 ug L−1in either intactor recolonized biofilms for 15 days in microcosms (mean time-weighted average concentration - TWACof 5.52 ± 0.74 ug L−1).At the end of the exposure period, alachlor effects were monitored by a combination of biomass descrip-tors (ash-free dry mass - AFDM, chlorophyll a), structural molecular fingerprinting (T-RFLP), carbonutilization spectra (Biolog) and diatom species composition. We found significant effects that in terms ofAFDM, alachlor inhibited growth of the intact phototrophic biofilms. No effect of alachlor was observedon diatom composition or functional and structural properties of the bacterial community regardless ofwhether they were intact or recolonized. The intact three-dimensional structure of the biofilm did notappear to confer protection from the effects of alachlor. Bacterial community structure and biomass levelof 4.4 weeks - intact phototrophic biofilms were significantly influenced by the biofilm maturation pro-cesses rather than alachlor exposure. The diatom communities which were largely composed of mobileand colonizer life-form populations were not affected by alachlor.This study showed that the effect of alachlor (at initial concentration of 10 ug L−1or mean TWAC of5.52 ± 0.74 ug L−1) is mainly limited to biomass reduction without apparent changes in the ecologicalsuccession trajectories of bacterial and diatom communities and suggested that carbon utilization spec-tra of the biofilm are not damaged resulting. These results confirmed the importance of consideringthe influence of maturation processes or community age when investigating herbicide effects. This isparticularly important with regard to the use of phototrophic biofilms as bio-indicators.