The spatial distributions of properties of biofilms have been investigated by using three different kinds of biofilms as test materials. Biofilms, cultured by laboratory-scale rotating drum biofilm reactors with synthetic wastewater, were first cut into 10 to 20 µm thick slices using a microtome, and then apportioned into samples representing 3 or 4 layers. The biofilm properties of each layer were investigated by measuring the densities, phospholipid concentrations, and AR18 dye adsorption abilities. The bacterial population distributions and the metabolically active bacterial distributions were studied by plate count methods or a MPN method, and the tetrazolium dye (INT) reduction method, respectively. Based on statistic evaluations, the micro-slicing technique, the procedure for analyzing phospholipid concentrations of biofilms, and the AR18 dye adsorption tests were suitable to be used in biofilm studies. It was found that the densities of biofilms in the bottom layers were 4 to 7 times higher than those in the top layers. For thick biofilms (thickness > 500 µm), the INT active bacteria decreased from 82-89% in the top layers to 5-11% in the bottom layers. The porosities of thick biofilms changed from 83-92% in the top layers to 56-64% in the bottom layers. For thin biofilms (thickness < 500 µm), the porosities of biofilms changed from 72-75% in the top layers to 35-44% in the bottom layers. Highly spatial distributions of bacterial populations, mean pore radius, and specific surface areas were also observed. As a result of these spatial distributions, the ratio of effective diffusivity to diffusivity in the bulk solution also shows a decrease with depth of the biofilm. Assuming biofilm properties are of a uniform distribution may be an over-simplified assumption, valid only in specific cases.