Dissolved organic matter (DOM) in groundwater influences water quality and fuels microbial metabolism, but its origins, bioavailability and chemical composition are poorly understood. The origins and concentrations of dissolved organic carbon (DOC) and bioavailable DOM were monitored during a long-term (2-year) study of groundwater in a fractured-rock aquifer in the Carolina slate belt. Surface precipitation was significantly correlated with groundwater concentrations of DOC, bioavailable DOM and chromophoric DOM, indicating strong hydrological connections between surface and ground waters. The physicochemical and biological processes shaping the concentrations and compositions of DOM during its passage through the soil column to the saturated zone are conceptualized in the regional chromatography model. The model provides a framework for linking hydrology with the processes affecting the transformation, remineralization and microbial production of DOM during passage through the soil column. Lignin-derived phenols were relatively depleted in groundwater DOM indicating substantial removal in the unsaturated zone, and optical properties of chromophoric DOM indicated lower molecular weight DOM in groundwater relative to surface water. The prevalence of glycine, γ-aminobutyric acid, and d-enantiomers of amino acids indicated the DOM was highly diagenetically altered. Bioassay experiments were used to establish DOC-normalized yields of amino acids as molecular indicators of DOM bioavailability in groundwater. A relatively small fraction (8 ± 4 %) of DOC in groundwater was bioavailable. The relatively high yields of specific d-enantiomers of amino acids indicated a substantial fraction (15–34 %) of groundwater DOC was of bacterial origin.