The fate of chemical of concern is not yet fully understood during treatment of impaired waters. The aim of this paper is to assess the impact of different organic-based fouling layers on the removal of a large range of trace organics. Both model and real water samples (mixed with trace organic contaminants at environmental concentration of 2 μg l−1) were used to simulate fouling in nanofiltration under controlled environment. The new and fouled membranes were systematically characterised for surface charge, hydrophobicity and roughness. It was observed that fouling generally reduced the membrane surface charge; however, the alterations of the membrane hydrophobicity and surface roughness were dependent on the foulants composition. The rejection of charged trace organics was observed to be improved due to the increased electrostatic repulsion by fouled membranes and the adsorption of the trace organic chemicals onto organic matters. On the other hand, the removal of nonionic compounds decreased when fouling occurred, due to the presence of cake enhanced concentration polarization. The fouling layer structure was found to play an important role in the rejection of the trace organic compounds.