This paper examines the effect of the fractional components of natural organic matter (NOM) in surface water on the fouling of microfiltration membranes. The results are shown for the NOM derived from the Mt. Zero drinking water reservoir located in the state of Victoria, but they are representative of the other two water reservoirs investigated in the full study. The NOM was concentrated and isolated into four fractions according to hydrophobicity and charge using non-functionalised and anionic resins. Filtration experiments were conducted in a stirred cell using 0.22 μm hydrophobic and surface-modified hydrophilic polyvinylidene fluoride (PVDF) membranes to examine the fouling effects by the NOM fractions on membranes of different hydrophobicity. The fouling rate and extent of fouling were considerably less for the hydrophilic membrane compared to the hydrophobic membrane, which was due to the reduced organic interactions (i.e., hydrophobic adsorption, charge interaction) between the NOM and the hydrophilic membrane. However, for both the hydrophilic and hydrophobic membranes, the order of the fouling potential of the four fractions was the same, namely hydrophilic neutral > hydrophobic acids > transphilic acids > hydrophilic charged. The explanation for the high fouling potential of the hydrophilic neutral fraction was attributed to the calcium and carbohydrates (mainly polysaccharides) in the NOM, which were found to concentrate in the hydrophilic neutral fraction. Fourier transform infrared spectroscopy (FTIR) spectra for the fouled hydrophobic PVDF and polypropylene membranes revealed some visible changes in the regions of Ar-O-Ar, C-C, C-N stretching and C-H rocking vibrations, indicating polysaccharides and aromatics being the important foulants on the hydrophobic membranes. The mechanism by which the hydrophilic neutral fraction caused severe membrane fouling was a combination of adsorption of polysaccharides on the membrane structure due to hydrophobic bonding of the non-polar segments of the polysaccharides, and electrostatic attraction interactions between the polysaccharide-Ca2+ complexes and the negatively charged membrane.