Natural organic matter (NOM) plays a significant role in the fouling of ultrafiltration membranes in drinking water treatment processes. For a better understanding of the interaction between fractional components of NOM and polysulfone (PS) ultrafiltration membranes used for drinking water treatment, fouling and especially the physically irreversible fouling of natural organic matter were investigated. Resin fractionation, fluorescence excitation–emission matrix (EEM) spectroscopy, fourier transform infrared spectroscopy (FTIR), contact angle and a scanning electron microscope (SEM) were employed to identify the potential foulants. The results showed that humic acid and fulvic acid of small size were likely to permeate the membrane, while the hydrophobic fraction of humic and fulvic acid and aromatic proteins tended to be rejected and retained. Organic compounds such as proteins, humic substances, and polysaccharide-like materials, were all detected in the fouling layer. The physically irreversible fouling of the PS membrane seemed to be mainly attributed to the hydrophobic fraction of humic substances.