Filtration using biological activated carbon (BAC) performs well in the removal of biodegradable dissolved organic carbon from water sources. The application of ozonation followed by up-flow BAC filtration has gained increasing attention in the world scale. In this study, a pilot-scale up-flow BAC filtration system was constructed for the treatment of polluted lake water. The operational results indicated that this BAC filtration system could effectively remove organic matter. Spatial heterogeneity of the microbial community structure inside the BAC filtration system was identified using bacterial 16S rRNA clone library analysis. A marked decrease of microbial diversity in the BAC filtration system was observed along the flow path. Alpha-proteobacteria, Gammaproteobacteria and Acidobacteria were found to be the major bacterial groups in the BAC filters. Moreover, Novosphingobium aromaticivorans-like microorganisms were detected. This work might add some new insights towards microbial communities in regards to BAC filtration for the treatment of drinking water.