Karst watersheds possess both diffuse and conduit flow and varying degrees of connectivity between surface and groundwater over spatial scales that result in complex hydrology and contaminant transport processes. The flow regime and surface-groundwater connection must be properly identified and characterized to improve management in karst watersheds with impaired water bodies, such as the Chapel Branch Creek (CBC), South Carolina watershed, which has a long-term sampling station presently listed on an EPA 303(d) list for phosphorous, pH, and nitrogen. Water from the carbonate limestone aquifer of the Santee Cave system and spring seeps in the CBC watershed were monitored to characterize dominant flow type and surface-groundwater connection by measuring dissolved calcium and magnesium, total suspended solids, volatile suspended solids, alkalinity, pH, specific conductance, and stable isotopes (d18O, d2H). These measurements indicated that the conduit flow to Santee Cave spring was recharged predominantly from diffuse flow, with a slow response of surface water infiltration to the conduit. Qualitative dye traces and stage elevation at Santee Cave spring and the adjacent Lake Marion (equal to the elevation of the flooded portion of CBC) also indicated a relation between fluctuating base level of the CBC reservoir-like embayment and elevation of the Santee Limestone karst aquifer at the spring. Methods described herein to characterize the flow type and surface-groundwater connection in the Santee Cave system can be applied not only to watershed management in the Chapel Branch Creek watershed, but also to the greater region where this carbonate limestone aquifer exists.