Biofilm is considered with respect to mass transport as an inert porous layer. The biofilm is developed in fresh water and in natural seawater on a disk electrode in gold. Its thickness is determined by considering the limiting diffusion current measured on this electrode for different rotation speeds. The same thickness is obtained when two different electrochemical tracers are used: Fe(CN)63− and oxygen which validates the technique and permits the use of oxygen naturally present in natural water as an electrochemical tracer. The dependence of the thickness on the rotation speed of the electrode shows the elastic behavior of the biofilm.