Organic matter in wastewater sampled from a swine waste-retention basin in Iowa was fractionated into 14 fractions on the basis of size (particulate, colloid, and dissolved); volatility; polarity (hydrophobic, transphilic, hydrophilic); acid, base, neutral characteristics; and precipitate or flocculates (floc) formation upon acidification. The compound-class composition of each of these fractions was determined by infrared and 13C-NMR spectral analyses. Volatile acids were the largest fraction with acetic acid being the major component of this fraction. The second most abundant fraction was fine particulate organic matter that consisted of bacterial cells that were subfractionated into extractable lipids consisting of straight chain fatty acids, peptidoglycans components of bacterial cell walls, and protein globulin components of cellular plasma. The large lipid content of the particulate fraction indicates that non-polar contaminants, such as certain pharmaceuticals added to swine feed, likely associate with the particulate fraction through partitioning interactions. Hydrocinnamic acid is a major component of the hydrophobic acid fraction, and its presence is an indication of anaerobic degradation of lignin originally present in swine feed. This is the first study to combine particulate organic matter with dissolved organic matter fractionation into a total organic matter fractionation and characterization.