A model, for evaluating the effect of porosity and volume fraction of extracellular polymeric substances (EPS) within multispecies biofilms on the effective diffusivity, is developed and experimentally validated, based on the extraction of EPS from intact biofilms. The amount of EPS in biofilms significantly affects the effective diffusivity. For biofilms with porosity of 77%-95% in the top layers and 54%-58% in the bottom layers, the value of D-e/D-w decreases from 0.52-0.83 in the top layers to 0.23-0.31 in the bottom layers. Generally, the effective diffusivity in the heterotrophic/autotrophic biofilms is slightly lower than that in the heterotrophic biofilms, due to the lower porosity in the heterotrophic/autotrophic biofilms.