The widespread research of graphene oxide (GO) membranes has revealed the need to investigate their long-term performance and associated biofouling behavior in practical water treatment conditions. In this study, we have evaluated the long-term operation of GO membranes from the perspective of filtration performance and biofilm characteristics, based on two representative GO membranes (V-GO and P-GO, fabricated by vacuum and pressure filtration, respectively) with different surface properties and a natural surface water, using a gravity driven membrane system. The results showed that the GO membranes were capable of achieving sustainable water purification over 45 days of operation, associated with a complete rejection for biopolymers and desirable removal for fluorescent organic matters. Furthermore, the V-GO membrane with a rougher surface, reduced hydrophilicity and higher sheet-edge exposure than that of the P-GO membrane formed a highly heterogeneous and porous biofilm with high permeability, despite its greater biofilm thickness and extracellular polymeric substances (EPS) contents. In contrast, the P-GO membrane biofilm exhibited a thin but dense structure, and therefore an increased hydraulic resistance (∼1.5 times greater than V-GO membrane). The microbial community analysis revealed that bacteria related to biofilm formation was richer in V-GO biofilm. While bacteria associated with the degradation of organic matters and the aggregation of microorganisms accounted for a greater proportion in P-GO biofilm. These factors were responsible for forming a thick biofilm for V-GO, and a thin but high resistance biofilm for P-GO membrane. Our findings highlight the sustainable water purification performance of the GO membranes, and the P-GO membrane can alleviate biofilm formation but not necessarily reduce biofouling during the long-term filtration, while this was opposite in case of the V-GO membrane.