The priming effect (PE), characterized as the enhanced microbial processing of bio-recalcitrant organic matter with the addition of labile substrates, has been hypothesized to moderate carbon (C) cycling in aquatic systems. In this study, aquatic PEs were evaluated through bacterial respiration and dissolved organic C consumption in incubations of water collected from three locations along the York River estuary. Incubations from White’s Landing on the Pamunkey River, a tidal freshwater tributary of the York, and from Croaker Landing in the middle of the estuary, displayed positive PEs when amended with labile C. In contrast, amended incubations from Gloucester Point, near the mouth of the estuary, displayed negative PEs, or reduced relative C metabolism, based on our calculations, This study provides empirical evidence for the occurrence of aquatic PEs and serves to elucidate how they may enhance or retard the processing and mineralization of organic C during transport to the ocean.