The interaction of shear stress with the biofilm leads to a dynamic deformation, which is related to the structural and material characteristics of biofilms. We show how optical coherence tomography can be used as an imaging technique to investigate the time-resolved deformation on the biofilm mesoscale as well as to estimate mechanical properties of the biofilm. For the first time time-resolved deformation from cross-sectional views of the inner biofilm structure could be shown. Changes in the biofilm structure and rheological properties were calculated from cross sections in real-time and time-lapsed measurements. Heterotrophic biofilms were grown in a flow cell set-up at low shear stress of (w)=0.01Pa. By applying higher shear stress elastic and viscoelastic behavior of biofilms were quantified. Deformation led to a change in biofilm conformation and allowed to estimate rheological properties. Assuming an ideal wall shear stress calculation, the shear modulus G=29.7 +/- 1.7Pa and the Young's modulus E=36.0 +/- 2.6Pa were estimated. Biotechnol. Bioeng. 2015;112: 1893-1905. (c) 2015 Wiley Periodicals, Inc.