The objective of this study was to develop a method to reliably and reproducibly assess the physical properties of in vitro multi-species plaque derived biofilms. A custom flow cell (FC) was designed to model oral cavity shear stresses on biofilms grown on hydroxyapatite (HA) discs. A finite-element program (ANSYS 13) modeled flow velocities and wall shear stresses on the interior 3D dimensions. For the experiment, 1% chlorhexidine (CHX), 5M urea, and a 1x phosphate-buffered saline (PBS) were flown through the FC simulating oral rinsing. Near infrared cross-polarization optical coherence tomography (CP-OCT) non-destructively imaged the fluid immersed biofilms in real time (20 frames/s). During low flow, the swell of the biofilm caused from 5M urea was quite pronounced increase in vertical dimension. Biofilms exposed to 1% CHX showed a slight collapse in the vertical dimension of the biofilm during low flow. During high flow/high sheer stress, the 5M urea solution effectively removed the biofilm, while both 1% CHX and 1x PBS did not remove biofilms even under high velocity/shear stress conditions. Biotechnol. Bioeng. 2016;113: 198-205. (c) 2015 Wiley Periodicals, Inc.