In natural environments, most microorganisms reside attached to a surface growing as a biofilm, which is a universal microbial strategy for their survival. As a result, several studies have focused on the role of the biofilm matrix (made of extracellular polymeric substances (EPS)) in tolerance to antimicrobials. However, few studies have focused directly on the characterization of EPS as a response to antibiotic stress. In this study, we analyzed the impact of trimethoprim (TMP) on the production and characteristics of EPS from four natural biofilm-producing river bacterial strains. Extraction and characterization of EPS were carried out at three concentrations of TMP. EPS properties were monitored using colorimetric tests, infrared spectroscopy and sugar composition analysis. For all strains, EPS quantity and chemistry changed starting at 0.1 mM TMP. The combined results suggest that environmental bacterial strains adapt their EPS production and chemical composition in response to antibiotic exposure. Bacteria may benefit from the change in EPS chemistry since it limits the penetration of TMP into the biofilm and thus protects the cells from the action of the antibiotic. Three main mechanisms are proposed: an increase in the proportion of (i) proteins and reactive functional groups, (ii) mannose and (iii) fatty acids. This study shows that EPS represents a key factor in antibiotic tolerance of bacteria via multiple mechanisms. Thus, this study broadens the discussion concerning antibiotic-resistance as it presents additional processes that may work in tune with genetically acquired resistance to enhance bacterial tolerance to antibiotics.