Polychlorinated biphenyls (PCBs) are compounds of significant interest due to high toxicity, persistence, long-range atmospheric transport, and bioaccumulation. These compounds can interact with components present in the environment, including dissolved organic matter (DOM) in soils and waters, thereby modifying its availability and movement. In this study, DOM was fractionated by ultrafiltration and characterized according to its hydrophobicity and hydrophilicity, then the interaction of a series of PCBs and different DOM fractions was evaluated. The DOM was collected from the surface waters of three sectors located along a river in the southern part of America. These sectors are subject to different anthropic activities, thus the DOM of sector 1, with the least anthropic influence, was mainly hydrophobic and with a high content of aromatic structures. In contrast, the DOM collected from sectors 2 and 3, where anthropic activity is highest, was slightly hydrophobic and hydrophilic, respectively. The DOM of these two sectors was mainly composed of low molecular weight macromolecules. These results revealed that more hydrophobic PCBs (i.e., 101, 118, 138, and 180) have a greater affinity to DOM with a higher molecular weight (i.e., >1 kDa). In turn, PCBs with lesser chlorination and hydrophobicity presented a greater affinity to DOM with a lower molecular weight. In conclusion, our study shows that the high molecular weight DOM is responsible for mobilizing PCBs with a high degree of chlorination.