There is consensus that temperature plays a major role in shaping microbial activity, but there are still questions as to how temperature influences different aspects of bacterioplankton carbon metabolism under different environmental conditions. We examined the temperature dependence of bacterioplankton carbon metabolism, whether this temperature dependence changes at different temperatures, and whether the relationship between temperature and carbon metabolism varies among estuarine sub-systems differing in their degree of enrichment. Two years of intensive sampling in a temperate estuary (Monie Bay, Chesapeake Bay, USA) revealed significant differences in the temperature dependence of bacterial production (BP) and respiration (BR), which drove a strong negative temperature response of bacterial growth efficiency (BGE). Accordingly, BGE was lower in summer (< 0.2) and higher in winter (> 0.5). For all measured metabolic processes, the most pronounced temperature response was observed at lower temperatures, with Q10 values generally 2-fold greater than in warmer waters. Despite significant differences in resource availability, both the temperature dependence and magnitude of BR and bacterioplankton carbon consumption (BCC) were remarkably similar among the 4 estuarine sub-systems. Although temperature dependencies of BP and BGE were also similar, their magnitude differed significantly, with highest values in the nutrient-enriched sub-system and lowest in the open bay. This pattern in carbon metabolism among sub- systems was present throughout the year and was confirmed by temperature manipulation experiments, suggesting the temperature effects on BP and BGE did not override the influence of resource availability. We conclude that temperature is the dominant factor regulating seasonality of BR and BCC in this system, whereas BP and BGE are influenced by both temperature and organic matter quality, with variation in the relative importance of each of these factors throughout the year.