Biofilms play an important role as sorbents in the process of distribution of pollutants in surface water systems. The sorption properties of biofilms will influence the kind and the amount of sorbed substances. The heterogeneity of biofilms provides different sorption sites which exhibit a different sorption preference and capacity. As dynamic systems, biofilms will respond physiologically to their environment. Thus, the sorption of one substance may lead to a change in the composition of the EPS or other biofilm components and further alter the original sorption properties. In this paper, the influence of toluene on a biofilm was investigated. As a suitable method, FTIR-ATR spectroscopy was applied. The method is non destructive and allows the observation of biofilm formation and behaviour on line and in situ. A biofilm was allowed to form in ATR flow-through cells. The test strain was genetically engineered and contained a bioluminescent reporter gene which was switched on when toluene was metabolized. Thus, the degree of toluene degradation activity could be observed with great sensitivity. The FTIR spectrometer contained three flow-through cells which could be operated in parallel: one was run with sterile medium only, one with medium and bacteria, and one with medium, bacteria and toluene. This arrangement allowed the discrimination of the biofilm response from other effects. The IR spectrum showed specific bands of proteins, polysaccharides, phosphoryl compounds and other groups of molecules. A significant increase of EPS-polysaccharide formation was observed at a toluene level of 5 mg L−1. At 15 mg L−1, significantly more carboxyl groups were formed. Thus, the effect of the lipophilic organic pollutant toluene increased the amount of negatively charged groups and, consequently, the sorption capacity for metal cations. This result indicates that biofilms respond in a complex manner to different sorbates and alter their environmental properties.