This study investigated the effect of the molecular weight cut-off (MWCO) of a dialysis membrane for the isolation of colloidal organic matter (COM) from surface water. Various dialysis membranes with different MWCO were used for the isolation of COM from Cebron reservoir natural organic matter (CRNOM). The COM included mainly amino sugars (3040%) and polysaccharides (3040%), with 23 of fatty acids over aromatic acids (F/A ratio), indicating that the COM were derived from microbial substances. As all the COM constituents were found to have similar characteristics, the MWCO of the dialysis membrane was not found to influence the COM characteristics.