Modification of conventional rapid sand filter into granular activated carbon–sand dual media filter has become increasingly attractive to drinking water producers in many Chinese urban regions. In this study, a pilot-scale dual media filter was constructed to monitor its performance. The operational results indicated that the dual media filter performed well both on nitrification and on reduction of chemical oxygen demand. Microbial community structures at different sampling sites were also compared using 16S ribosomal ribonucleic acid gene clone library analysis to identify the spatial heterogeneity the filter. Clone library analysis illustrated the difference of microbial community structure and an increase of microbial diversity along the filter depth. Moreover, potential pathogens from genera Chromobacterium and Sphingomonas were detected in the filter samples, which deserved further serious consideration in downstream treatment such as disinfection. These findings also provided an illustration of the utility of molecular techniques to discover microbial community change and potential pathogens in the drinking water biofilters.