Composite membranes with a cross-linked chitosan active layer (CS-membrane) on a poly(ether sulfone) (PES) support layer surface-functionalized with acyl chloride groups were fabricated as nanofilters. The support layers composed of PES and PES functionalized with amine groups (PES-Am) were surface-functionalized with acyl chloride groups (membrane-COCl). The membrane-COCl reacted further with amine groups in chitosan to form CS-membrane. The CS-membrane exhibited increased salt removal rates, antibacterial activity, and a decrease in water flux with increasing PES-Am content up to 20 wt % in the support layer. The CS-membrane formed on the support layers containing 20 wt % PES-Am and a commercial NF membrane showed almost the same MgSO4 rejection (97.5%), whereas the former had about 1.4 times higher water flux than the latter. Consequently, NF membranes having better water flux, fouling resistance, and antibacterial activity than commercial NF membrane were fabricated without reducing salt rejection by assembling a cross-linked chitosan layer on a PES support layer surface-functionalized with acyl chloride groups.