The diffusion coefficients of three different chemical species in naturally grown, heterotrophic biofilms have been measured and a simple model of mass transfer in a biofilm matrix is developed. The mechanical structure of the biofilm matrix reduces the molecular diffusion to about 50–80% of the value in pure water. Depending on the roughness of the biofilm surface and the flow conditions eddy diffusion increased the mass transfer into the biofilm near the surface. The influence of the diffusion potential and the donnan potential on ions have been evaluated by comparing the diffusion coefficients of a positively and a negatively charged ion and a neutral molecule in experiments with different background electrolyte concentrations. Mass transfer effects by electrostatic forces are negligible at the ionic strength of waste water and tap water.