An experimental study was undertaken to highlight the potential ecotoxicological impact of the herbicide diuron on biofilms during flooding events in a small river (Morcille) in the Beaujolais vineyard area (France). We investigated the responses of chronically-contaminated biofilms exposed to short-term pulses (3 h) of diuron. Biofilms were grown in indoor microcosms that were either non-contaminated or exposed to low-level chronic contamination, and was not exposed, or exposed to single or double pulses of two environmental concentrations (7 and 14 µg L-1) of diuron. Exposure to pollution and its impact on biofilms were assessed by measuring pesticide concentrations in biofilms, biomass parameters (chl a, AFDW), community structure (using 18S and 16S rDNA gene analysis by DGGE, and HPLC pigment analysis to target eukaryotes, bacteria and photoautotrophs respectively) and by performing a physiological test. Control biofilms displayed very low diuron concentrations, the herbicide being found in the contaminated biofilms. Nevertheless, diuron concentrations were not higher in the pulsed biofilms than in the non-pulsed ones. AFDW and chl a in-vivo fluorescence increased in both microcosms during the experiment and biomass was higher in chronically-exposed biofilms than in control ones. The impact on biomass was higher for the control double-pulsed biofilms than forf the non-pulsed ones. Carbon incorporation by the chronically-exposed biofilms was greater during the first 28 days of growth than during the first 28 days of growth than by the control biofilms. Both single and double pulses inhibited carbon incorporation of all biofilm communities, especially of the control ones. Short-term inhibition of photosynthesis was never significantly different in exposed and non-exposed biofilms. Few differences in the pigment structure were found between chronically-exposed biofilms and control biofilms, but pulses impacted on the pigment structure of all biofilm communities. Bacterial structural differences were observed between single-pulsed and non-pulsed biofilms, but not between double-pulsed and non-pulsed biofilms. The different pulses affected the eukaryotic community structures of the control biofilms, but not of the chronically-exposed ones. Unlike the bacterial communities, the control eukaryotic communities were structurally different from the chronically-exposed ones. This preliminary experimental study indicates that exposure to environmental concentrations and exposure of diuron can have measurable effects on small river biofilm communities. The effects of a pulsed acute exposure to diuron on biofilms depended on whether the biofilms had previously been exposed to the same stressors or not.