Numerous studies have demonstrated the worldwide occurrence of pharmaceutical contamination in aquatic environments, in which they can affect microbial periphytic communities. Recent monitoring in a small river located in Lake Bourget watershed (Tillet River, Savoie, France) showed the accumulation of pharmaceuticals in its downtream urban section. In this context, we sought to (i) assess if this chronic exposure to contamination can cause an increase in the tolerance of microbial communities to a set of pharmaceutical substances (‘pollution induced community tolerance’ concept, PICT) and (ii) study the tolerance dynamics (tolerance acquisition, or loss) according to exposure level changes. To reach these objectives, we carried out an in situ translocation study in the Tillet River, considering an upstream (reference) and a downstream (contaminated) site. After a 4-week growth phase in each site, periphytic biofilms were transferred between the sites to simulate, during 6 weeks, a restoration (down- to upstream) or deterioration (up- to downstream) of the chemical water quality. Water contamination was monitored using polar organic chemical integrative samplers (POCIS), and biofilm community tolerance was assessed using a PICT approach. Acute toxicity tests were performed on biofilms sampled every 2 weeks, using 7 pharmaceuticals tested individually (ofloxacin, atenolol, diclofenac, paracetamol, erythromycin, sulfamethoxazole, sulfamethazine) and measuring the following biological activities: β-glucosidase, photosynthesis, or growth. Our results confirmed the expected increase in pharmaceutical concentrations in surface water from up- to downstream. In parallel, an increased biofilm tolerance mainly towards diclofenac and atenolol was observed at the downstream station, from the toxicity tests on photosynthesis. The translocation approach also revealed an increase of tolerance levels in communities transferred downstream, and a decrease of tolerance levels in communities transferred upstream, suggesting resilience processes. This work demonstrates the relevance of the PICT approach for in situ diagnosis of the impact of pharmaceuticals on natural microbial communities.