Advanced characterization techniques of organic matter, including bulk organic characterization, size-exclusion chromatography, three-dimensional excitation–emission matrix, Fourier transform infrared spectroscopy, and fractionations using Amberlite XAD-8/4 resins, were used to investigate differences and similarities in the physicochemical properties of four different organic matter, namely algogenic organic matter (AOM), bacterial organic matter (BOM), Suwanee River humic acids (SRHA) and Suwanee River fulvic acids (SRFA). From the comparison of characteristics of the AOM, BOM, SRHA, and SRFA, it was identified that the specific UV absorbance, molar ratio of organic nitrogen to organic carbon, molecular weight, fluorescence characteristics, functional group compositions, and relative hydrophobicity/hydrophilicity of all the tested organic matter were considerably different from their sources. The SRHA and SRFA were mainly composed of hydrophobic fractions while the AOM and BOM included more hydrophilic fractions than the SRHA and SRFA due to the alcohol and amide functional groups. This indicated that the origin of organic matter in natural waters can be predicted by their physicochemical characteristics, and the source identification of organic matter provides a better understanding of the interactions between the origin of organic matter and water treatment processes (e.g., coagulation and membrane filtration).