Direct measurement of unsteady-state variation of oxygen concentrations inside deactivated biofilm at intervals of 100 μm was conducted with oxygen microelectrodes. The diffusivity of each layer was estimated using an explicit finite-difference method. The results show that the distribution of the biofilm effective oxygen diffusivity varies from 25% Dw at the substratum of the biofilm to 90% Dw at the surface of the biofilm. This information provides experimental evidence necessary for biofilm modelling that could not be approached in the past, and will create a new dimension for evaluation of biofilm processes.