One of the main factors affecting membrane fouling in MBRs is operational conditions. In this study the influence of aeration rate, filtration mode, and SRT on hollow fiber membrane fouling was investigated using a triple fouling layers perspective. The sludge microbial population distribution was also determined by PCR method. Through various applied operational scenarios the optimal conditions were: aeration rate of 15 LPM; relaxation mode with 40s duration and 8 min. interval; and SRT of 30 days. The similarity between SMP variations in triple fouling layers with its corresponding hydraulic resistance confirmed the effect of SMP on membrane fouling. Among three fouling fractions, the upper (rinsed) layer found to have the most effect on membrane fouling which implies the critical role of aeration, but as for multilateral effects of aeration, the optimal aeration rate should be determined more precisely. Relaxation interval was more effective than its duration for fouling control. SRT variations in addition to influencing the amount of SMP, also affect on the structure of these material. At longer SRTs (20, 30 days) a greater percentage of SMP could penetrate into the membrane pores and for shorter SRTs they accumulate more on membrane surface. Results showed that there is a very good correlation between total hydraulic resistance (Log R) and protein to carbohydrate ratio at the rinsed layer (P1/C1). Considering significant effects of aeration and SRT conditions on this ratio (according to data), it is very determinative to apply the optimal aeration and SRT conditions.