The composition and metabolic capability of bacterioplankton communities were examined over seasonal and spatial gradients and related to the source, composition, and quantity of dissolved organic matter (DOM) in the blackwater estuary Winyah Bay, Georgetown County, SC, USA and its tributary rivers. Bacterial community composition (BCC) was measured by terminal restriction fragment length polymorphism, and bacterial metabolic capability (BMC) was measured by defined substrate utilization patterns (Biolog GN2 plates). Spatial patterns were not important, despite the anticipated watershed effects and the well-documented influence of salinity gradients on estuarine bacterioplankton, but DOM, BCC, and BMC all showed varying degrees of temporal patterns; DOM-based groupings differentiated BCC samples better than spatiotemporal categories, but not BMC. BCC was closely related to properties describing DOM composition, particularly those related to DOM source (i.e., cypress swamps vs. in situ phytoplankton production, indicated by chlorophyll a, colored DOM spectral slope, alpha 355/dissolved organic carbon (DOC), and DOC concentration), and to associated physicochemical variables, such as temperature, pH, and salinity. BMC was more strongly related to abiotic factors, such as temperature and dissolved nutrients, as well as to chlorophyll a and percent bioavailable DOC. In contrast with previous studies, BCC and BMC were significantly correlated in this highly heterotrophic estuary, suggesting that DOM source variability may select for specialist phylotypes above a background of generalists. This study, therefore, supports a causative pathway from DOM to BMC to BCC while suggesting that BCC and BMC may be simultaneously influenced by different suites of DOM characteristics and physicochemical parameters.