A pure culture of Pseudomonas putida was grown as a film on carbon particles in a differential fluidized bed biofilm reactor, a new experimental system for the application of diffusion-reaction models. In the active biofilm, effective diffusion coefficients of the essential substrates—phenol and oxygen—were simultaneously calculated. The multisubstrate biokinetic expression used in the model solution was derived by nonlinear regression analysis of the data of continuous system fermenter experiments. The determined biokinetic equations were utilized to solve the diffusion-reaction model for effective diffusion coefficients in the active biofilm. The ratio of the evaluated effective diffusion coefficient through active biofilm to that of water varied between 17–44% and 9–24% for phenol and oxygen, respectively, for different biofilm densities. Results of the study showed a fair agreement with the literature at low biofilm densities.