Particle size distribution (PSD)-based fractionation, an alternative methodology in wastewater characterization, was employed to gather detailed information on the organic matter content of a raw olive mill wastewater (OMW) and to investigate the changes brought about in this characteristic after application of certain chemical treatment alternatives, namely, lime precipitation-coagulation, Fenton oxidation, and electrocoagulation. PSD-based analysis of the untreated OMW demonstrated that more than two-thirds of its organic carbon content, measured as chemical oxygen demand (COD) (69%), total organic carbon (TOC) (74%), as well as antioxidant activity (AOA) (74%) caused by the polyphenolic carbon fraction, was at the soluble range (< 2 nm). Treatability experiments, with maximum overall removal efficiencies below 50% even under optimized conditions, indicated that none of the applied chemical pretreatment methods was sufficiently effective in removing the organics from the investigated OMW. Nonetheless, PSD-based fractionation of the pretreated samples provided an insight about the treatment alternatives and the size fractions where they performed better. Electrocoagulation was capable of removing particulate and colloidal matter and provided a relatively higher overall performance with 23, 20, and 34% decreases in COD, TOC, and polyphenol contents of the OMW, respectively. Fenton process, on the other hand, showed a lower overall performance in terms of COD and TOC removal ( 17 and 15%, respectively), yet it was significantly effective on the soluble fraction, and thus more successful in removing the polyphenols mostly originating from the soluble range, with an overall efficiency of 42%. Based on these observations, PSD analysis was suggested as a useful tool to perform detailed wastewater characterization, as well as to differentiate between specific impacts of different pretreatment processes and help choose between alternatives, especially when a particular pollutant is targeted.