Nanofiltration (NF) membrane fouling by surface waters is still an unclear and unpredictable topic due to the heterogeneity and variability of natural organic matter (NOM) in waters. The objective of this study was to identify the key water quality parameter/s responsible for NF membrane fouling. Six representative surface waters were systematically investigated in terms of the water quality characteristics and their fouling potential on a polyamide NF membrane. The results showed that these waters exhibited significant discrepancies in DOC, Ca2+, composition, and chemical characteristics of NOM. Removal performance and membrane fouling analysis indicated a preferential rejection of hydrophobic over hydrophilic NOM, and the hydrophobicity of NOM was positively correlated with reversible fouling. The biopolymer content exhibited a strong positive correlation to the total membrane fouling and irreversible fouling, indicating that it could be used as a surrogate of NF fouling behaviors by surface waters. Ca2+ and humic substances contents could not explain the membrane fouling due to their insignificant correlation with membrane fouling. However, the proportion of high molecular weight component in humic substances (%-HMW) exhibited a negative correlation with the total membrane fouling and irreversible fouling, suggesting that the HMW component has a positive effect on NF membrane fouling prevention. A reasonable explanation is that the HMW component is preferentially deposited on the membrane surface, forming a ‘controlling layer’ that prevents low MW organics from entering and blocking the membrane pores.