Dissolved organic matter (DOM) in groundwater and surface water samples from the Florida coastal Everglades were studied using excitation-emission matrix fluorescence modeled through parallel factor analysis (EEM-PARAFAC). DOM in both surface and groundwater from the eastern Everglades S332 basin reflected a terrestrial-derived fingerprint through dominantly higher abundances of humic-like PARAFAC components. In contrast, surface water DOM from northeastern Florida Bay featured a microbial-derived DOM signature based on the higher abundance of microbial humic-like and protein-like components consistent with its marine source. Surprisingly, groundwater DOM from northeastern Florida Bay reflected a terrestrial-derived source except for samples from central Florida Bay well, which mirrored a combination of terrestrial and marine end-member origin. Furthermore, surface water and groundwater displayed effects of different degradation pathways such as photodegradation and biodegradation as exemplified by two PARAFAC components seemingly indicative of such degradation processes. Finally, Principal Component Analysis of the EEM-PARAFAC data was able to distinguish and classify most of the samples according to DOM origins and degradation processes experienced, except for a small overlap of S332 surface water and groundwater, implying rather active surface-to-ground water interaction in some sites particularly during the rainy season. This study highlights that EEM-PARAFAC could be used successfully to trace and differentiate DOM from diverse sources across both horizontal and vertical flow profiles, and as such could be a convenient and useful tool for the better understanding of hydrological interactions and carbon biogeochemical cycling.