The removal of natural organic matter (NOM) in term of CODMn by up-flow biologically activated carbon filter (UBACF) and down-flow biologically activated carbon filter (DBACF) was investigated in a pilot-scale test. The impacts of the molecular weight distribution of NOM on its degradation by the UBACF and DBACF were evaluated. The relationship between biodegradation and the microbial properties in the UBACF and DBACF were approached as well. The feed water of the UBACF and DBACF were pumped from the effluent of the rapid sand filtration (RSF) of Chengnan Drinking Water Treatment Plant (CDWTP), Huaian, Jiangsu Province, China. When the adsorption was the dominant mechanism of NOM removal at the initial stage of operation, the CODMn removal efficiency by the UBACF was lower than the DBACF. However, with the microbes gradually accumulated and biofilm formed, the removal of CODMn by the UBACF increased correspondingly to 25.3%, at the steady-state operation and was approximately 10% higher than that by the DBACF. Heterotrophy plate count (HPC) in the finished water of the UBACF was observed 30% higher than that of the DBACF. The UBACF effluent had higher concentration of detached bacteria whereas the DBACF harbored more attached biomass. The highest attached biomass concentration of the UBACF was found in the middle of the GAC bed. On the contrary, the highest attached biomass concentration of the DBACF was found on the top of the GAC bed. Furthermore, a total of 9479 reads by pyrosequencing was obtained from samples of the UBACF and DBACF effluents. The UBACF effluent had a more diverse microbial community and more even distribution of species than the DBACF effluent did. Alphaproteobacteria and Betaproteobacteria were the dominant groups in the finished water of the UBACF and DBACF. The higher organic matter removal by the UBACF was attributed to the presence of its higher biologically activity.