Recovery of bacterial and eukaryotic communities in biofilms naturally grown on stones was studied for 9 weeks after transferring them from a pesticide polluted downstream site of the river Morcille (Beaujolais, France) to a non-contaminated upstream site. Site-specific periphyton present on stones at both the down- and the upstream sampling site were collected to analyze the site-specific colonization. Throughout the experiment, structural and functional parameters were analyzed for the periphyton transferred and for the site-specific up- and downstream periphyton. Comparison between these three communities allowed quantifying recovery of the transferred one. Denaturing Gradient Gel Electrophoresis of PCR-amplified 16S and 18S rRNA gene fragments were used to assess prokaryotic and eukaryotic microbial community composition, respectively. Microscopy counts allowed characterizing the diatom taxa abundances. The sensitivity of the microalgal communities towards diuron and copper was investigated at the laboratory by short-term photosynthesis inhibition assays. The functional reaction of the bacterial communities towards copper was assessed by short-term respiration inhibition assays. The structure of transferred eukaryotic, bacterial and diatom communities was more similar to the structure of the downstream communities than to upstream ones even after 9 weeks acclimatization in particular for the bacterial community. In the same way, the community tolerance towards diuron and copper, as estimated by the EC50 values, was intermediate for the transferred biofilms compared to the local up- or downstream biofilm, even after 9 weeks of acclimatization. These results strongly suggest slow recovery, likely to be linked to long lasting exposure of pesticides and in particular copper adsorbed to the biofilm matrices and to the toughness for pioneer microorganisms to invade mature biofilms.