The role of chemical and physical interactions in natural organic matter (NOM) fouling of nanofiltration membranes is systematically investigated, Results of fouling experiments with three humic acids demonstrate that membrane fouling increases with increasing electrolyte (NaCl) concentration, decreasing solution pH, and addition of divalent cations (Ca2+). At fixed solution ionic strength, the presence of calcium ions, at concentrations typical of those found in natural waters, has a marked effect on membrane fouling. Divalent cations interact specifically with humic carboxyl functional groups and, thus, substantially reduce humic charge and the electrostatic repulsion between humic macromolecules. Reduced NOM interchain repulsion results in increased NOM deposition on the membrane surface and formation of a densely packed fouling layer. In addition to the aforementioned chemical effects, results show that NOM fouling rate increases substantially with increasing initial permeation rate. It is demonstrated that the rate of fouling is controlled by an interplay between permeation drag and electrostatic double layer repulsion; that is, NOM fouling of NF membranes involves interrelationship (coupling) between physical and chemical interactions. The addition of a strong chelating agent (EDTA) to feed water reduces NOM fouling significantly by removing free and NOM-complexed calcium ions. EDTA treatment of NOM-fouled membranes also improves the cleaning efficiency dramatically by disrupting the fouling layer structure through a ligand exchange reaction between EDTA and NOM-calcium complexes.