It is of practical significance to explore the fouling mechanism of nanofiltration membrane caused by residual natural organics left over after ultrafiltration unit. This paper systematically investigates nanofiltration membrane fouling behaviors induced by preserved humic acid components after ultrafiltration filtration (PHACUF) in the existence of Mg2+/Ca2+ ion from micro- and macro-level analyses. Those similarities and disparities in the roles of polyethersulfone or polyamide active layer to these issues were also studied comparatively. The results showed that there existed unneglectable standard blocking for polyethersulfone membrane when permeating bare PHACUF solution. However, with the addition of divalent cations, the main fouling mechanism changed to intermediate blocking. Fewer Mg2+ ions could fully link with the carboxylic groups of PHACUF molecules, leading to larger and more dense flocs by the “bridging” effect and the aggravated nanofiltration membrane fouling with more compact layer structure, especially for more hydrophobic polyethersulfone membrane. It could be inferred that in Mg2+/Ca2+ environment, actually, PHACUF-caused nanofiltration membrane fouling was mainly formed and constituted by hydrophobic acids and neutral fractions with much higher both hydrophobicity and content percentage, whether for the membrane-foulant interaction in the early stage or the foulant-foulant interaction in subsequent permeation stage.