The influence of an aerobic fixed-biofilm activity, microbial ecosystem and mass transfer with respect to HRT variation in a BNR (biological nutrient removal) system has been investigated in this study. The process used in this study was an anoxic (1)/aerobic (1)/anoxic (2)/aerobic (2) system. The study was demonstrated by several kinds of techniques such as INT-dehydrogenase activity (DHA), INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride), DAPI (4',6'-diamidino-2-phenylindole hydrochloride), and microelectrode. The study used by synthetic wastewater and HRT variation demonstrated that the DHA activity, density and heterotrophs/autotrophs ratio increased, as the HRT decreased from 8 hr to 4 hr. In comparing two aerobic reactors in fixed-biofilm process, the first aerobic reactor of the higher C/N ratio showed higher heterotrophs/autotrophs ratio and microbial activity than the second aerobic reactor. It was therefore concluded that the heterotrophs/autotrophs ratio and microbial activity were a greater influence on the first aerobic reactor, as organic loading rate was increased by HRT variation.