Wastewater effluent organic matter (EfOM) was isolated into different fractions including colloids, and hydrophobic (HPO) and transphilic (TPI) fractions. The EfOM isolates were characterized by different techniques, for example, size exclusion chromatography (SEC) with on-line UVA and DOC detectors, Fourier transform infrared (FTIR), specific UVA (SUVA), and total sugars analysis. The colloidal fraction is primarily composed of polysaccharides, proteins, and/or aminosugars, providing a hydrophilic character. The HPO and TPI fractions possessed characteristics of humic substances, i.e. high aromaticity and carboxylic functional groups. A superimposition of the EfOM isolates reflects characteristics of bulk wastewater effluents, consisting of refractory natural organic matter (NOM) conveyed from the drinking water source and soluble microbial products (SMP) derived during biological processes of wastewater treatment. Each EfOM isolate exhibited different characteristics in fouling of NF and UF membranes due to their distinct characters. The colloidal fraction showed high flux decline and fouling on NF and UF membranes primarily due to the effects of pore blockage. The HPO and TPI fractions exhibited less fouling and flux decline than the colloids due to their molecular size as well as electrostatic repulsion between organic acids and the membrane surface. However, hydrophobic interactions play a significant role with hydrophobic membranes, causing a reduction of permeate flux. Membrane autopsies using FTIR identified functional groups of organic foulants, supporting the evidence of flux decline by each EfOM isolate. Polysaccharides and/or aminosugars from the colloids in wastewater effluent were found to play an important role in fouling of NF and UF membranes.