In this study, a pure culture of Zoogloea ramigera was film-wise grown on activated carbon particles in a differential fluidized bed biofilm reactor (DFBBR). In biofilm, effective diffusion coefficients of the essential substrates; glucose, ammonium chloride and dissolved oxygen were simultaneously calculated. The multisubstrate biokinetic expression used to calculate effective diffusion coefficients was derived by nonlinear regression analysis of the data of continuous fermenter experiments. This equation was utilized to solve diffusion & reaction model for effective diffusion coefficients in active biofilm. The results of the model solution revealed that the effective diffusion coefficient for all three substrates through the biofilm decreased with increasing biofilm density.