Environmental and chemical properties were investigated in water samples collected at sites in Kings Bay, Florida, within its springshed, and in the adjacent Crystal River, in an attempt to discover the cause of observed increases in nitrogen and nuisance filamentous algae over the past 40 yr. Colored dissolved organic matter (CDOM) was characterized by excitation emission matrix and absorption spectroscopy. Subsequent parallel factor analysis (PARAFAC) identified five recurring peaks: two terrestrial humic (C-type), two marine humic (M-type), and one protein-like component. Optical and environmental parameters used for discriminant analysis included fluorescence maximum intensity (Fmax), emission maximum (Emmax), absorption coefficient [aCDOM (λ)], spectral slope (S), PARAFAC components, total nitrogen (TN), dissolved inorganic nitrogen, dissolved organic nitrogen, ratio of TN to Fmax (TN : F), total phosphate, and salinity. Canonical analysis of principal (CAP) coordinates indicated that 82% of the samples could be accurately categorized on the basis of their site of origin. Northern Kings Bay sites were characterized by a protein-like component associated with wastewater, as well as DIN concentrations in excess of the United States Environmental Protection Agency limit of 0.35 mg NO3 L−1. Two distinct M-type humic components were identified: partially photobleached component that was found in the brackish water and unbleached component found in meteoric groundwater. We propose that the latter can be utilized as a groundwater marker. Our results provide a model by which meteoric and subterranean estuary groundwater sources may be uncovered through the use of multiple environmental parameters combined with PARAFAC components in multivariate analyses.