The mobility of water in intact biofilms was measured with pulsed field gradient nuclear magnetic resonance (PFG-NMR) and used to characterise their diffusive properties. The results obtained with several well-defined systems, viz. pure water, agar, and agar containing inert particles or active bacteria were compared to glucose diffusion coefficients measured with microelectrodes and those calculated utilising theoretical diffusion models. A good correspondence was observed indicating that PFG-NMR should also enable the measurement of diffusion coefficients in heterogeneous biological systems. Diffusion coefficients of several types of natural biofilms were measured as well and these results were related to the physical biofilm characteristics. The values had a high accuracy and reflected the properties of a sample of ca. 100 biofilms, while nonuniformity or non-geometrical shapes did not negatively influence the results. The monitored PFG-NMR signal contains supplementary information on e.g. cell fraction or spatial organisation but quantitative analysis was not yet possible.