This work aimed to determine the effect of organic foulants and fouling behavior on separation of multi-ions for nanofiltration (NF) membrane. Three model organic foulants (bovine serum albumin, lysozyme, and humic acid) were selected and studied in depth. The rejections of mono- and di-anions by virgin membrane varied with different organic foulants because of foulant-enhanced co-ion competition. Subsequent multi-ions separations by fouled membranes were different due to the zeta potential and structure of the fouling cake. In contrast to the single protein system, Cl- and rejection was reduced by HA fouled membrane. However, with added proteins in the HA solution, mixed systems experienced decreasing Cl- rejection and increasing rejection. In addition, solution chemistry (solution pH, ionic strength, and Ca2+) had marked effect on the cake layer composition, likely due to the changing interaction between foulant and foulant, as well as between foulant and NF membrane. The suitable foulant cake structure could result enhanced in multi-ions separation. The synergistic mechanisms of organic foulants and cake were analyzed by calculated cake hydraulic resistance and zeta potential of fouled membranes.