Many industrial processes are susceptible to biofouling. The thickness and structure of such biofilms are key factors in the design of effective cleaning strategies. A novel method based on fluid dynamic gauging has been developed for measuring the thickness and the shear stress needed for removal of the biofilms formed on cylindrical surfaces. The device operates with the test cylinder immersed in liquid: liquid is withdrawn or ejected from a nozzle located near the biofilm surface. There is no net change of liquid volume, making it ideal for sterile and aseptic operation and for studies using valuable liquids. Biofilm removal may also be tested by using appropriate hydrodynamic conditions. Calibration tests using ejection and suction flows in the laminar regime (Reynolds number around 100) indicated a measurement accuracy of 19 μm and showed good agreement with computational fluid dynamics simulations. The device was commissioned in tests on Pseudomonas fluorescens biofilms formed on high density polyethylene (HDPE) and stainless steel (SS) cylinders of diameter 25 mm under conditions of mild shear stress (around 2 Pa in these tests). The biofilm thickness was not uniform to the eye and measurements made over the surface of the test cylinders confirmed this: layer thicknesses ranged from effectively zero to 200 μm. The biofilms formed on HDPE were thicker than those formed on SS.