The anchoring biofilm layer is expected to exhibit a different response to environmental stresses than for portions in the bulk, due to the protection from other strata and the proximity to the attachment surface. The effect of hydrodynamic stress on surface-adhered biofilm layers was tested using a specially designed microfluidic bio flow cell with an embedded three-electrode detection system. In situ electrochemical impedance spectroscopy (EIS) measurements of biocapacitance and bioresistance of Pseudomonas sp. biofilms were conducted during the growth phase and under different shear flow conditions with verification by other surface sensitive techniques. Distinct, but reversible changes to the amount of biofilm and its structure at the attachment surface were observed during the application of elevated shear stress. In contrast, regular microscopy revealed permanent distortion to the biofilm bulk, in the form of streamers and ripples. Following the application of extreme shear stresses, complete removal of significant portions of biofilm outer layers occurred, but this did not change the measured quantity of biofilm at the electrode attachment surface. The structure of the remaining biofilm, however, appeared to be modified and susceptible to further changes following application of shear stress directly to the unprotected biofilm layers at the attachment surface.