The removal dynamics of biodegradable dissolved organic carbon (BDOC) and natural organic matter (NOM) polarity fractions at a water treatment plant (WTP) in South Africa was studied using UV-Vis absorbance, fluorescence excitation-emission matrix, and two-dimensional synchronous fluorescence spectroscopy (SFS). This study gave insights into the transformation of NOM due to treatment processes. The objectives of the study were: (i) to use chemometric analysis and two-dimensional SFS correlations to investigate the evolution of NOM arising from treatment processes, and (ii) to access the chemical profile dynamics of polarity and BDOC fractions throughout the treatment train. The UV254 absorbance, which indicates aromaticity, reduced by 45% along the WTP. Gaussian fitting of UV-Vis data showed a decreasing trend in intensity and number of bands along the treatment process. The removal efficiency of NOM components followed the order: humic-like (HL) > tyrosine-like (TYL) > fulvic-like (FL) > tryptophan-like (TPL) > microbial-like (MBL). At the source, the relative distribution of the hydrophobic (HPO), hydrophilic (HPI), and transphilic (TPI) fractions was 45%, 31%, and 24%, respectively. The HPI was recalcitrant to treatment, and the TYL component of the HPI fraction was conjectured to be a disinfection byproduct limiting reagent. The HL and FL components of the BDOC fraction were the major substrates for bacterial growth. According to two-dimensional-SFS correlation, TYL, TPL, and MBL varied concurrently across the treatment stages. Used for the first time in South Africa, the robustness of a multi-dimensional approach of optical methods coupled with chemometric tools for the assessment of the fate of NOM along the treatment processes was revealed by this study.