We describe the use of Monie Bay as a natural experiment to evaluate the effect of system-level nutrient enrichment on natural bacterioplankton communities. Monie Bay, a component of the Chesapeake Bay National Estuarine Research Reserve System, is a subestuary of the Chesapeake Bay, consisting of a shallow semienclosed bay and three tidally influenced creeks varying in their agricultural land use and freshwater inputs. As part of a 2-year study in this system, we identified distinct spatial and seasonal patterns in ambient nutrient concentrations, salinity, and source and quantity of organic matter that were related to differences in agricultural practices and watershed characteristics among the three tidal creeks. Principal components analysis identified freshwater delivery of nutrients and temperature as key factors driving the overall variability of this system. Despite significant variability in nutrient concentrations and bacterioplankton production (BP) throughout the year, we observed persistent response of bacterioplankton to nutrient enrichment, as evidenced by a comparison of 2-year averages in agriculturally developed Little Monie Creek (LMC) relative to the undeveloped Little Creek (LC), and by a comparison of the nutrient-enriched upper estuary of LMC to sites nearer the open bay. Bacterioplankton responded positively to pulsed nutrient availability, with elevated rates of BP associated with agriculturally derived nutrient inputs to the Monie Bay system. Freshwater inputs play an important role in mediating the response of bacterioplankton to nutrient enrichment, as evidenced by relatively low estimates of BP in the freshwater-dominated, agriculturally developed Monie Creek. This response is attributed to changes in organic matter quality in the system and the direct effect of salinity on bacterioplankton community metabolism.