The bioactivity was enhanced by preozonation under low temperature conditions. Higher level of BDOC/AOC and DO may enhance the nitrifying performance. High level of biodiversity and bioactivity may help maintain the stability of filters. Water quality and DO could selectively enhance different microbial communities. The combination of preozonation and subsequent biological granular activated carbon (O /BAC) filtration is well known as a promising method for the removal of many pollutants. Temperature and nutrients are the dominant factors in external conditions to influence the biological communities. To explore the influence of preozonation under low temperature, the factors such as dissolved oxygen (DO), dissolved organic carbon (DOC) and NH -N were analyzed from the sampling ports every week; triphenyl tetrazolium chloride-dehydrogenase activity (TTC-DHA) and the nitrifying activity were detected along the bed height of biofilter at four levels (10, 40, 70 and 110 cm) on the 90th, 110th, and 130th day; microbial community, based on 16S rRNA gene-denaturing gradient gel electrophoresis (DGGE), was monitored on the 130th day of the operation. The observed microbial property showed that preozonation had a positive influence on bioactivity, biomass and nitrifying activity. Community analysis showed no significant difference on the biodiversity of nitrifying bacteria between the parallel filters in the inlet end based on the method employed. This result showed that biofilters’ performance is not correlated well with microbial biodiversity. The elevated functionality in O /BAC filters can be a result of increased microbial activity, which was promoted by preozonation.