Aquatic microbial responses to changes in the amount and composition of dissolved organic carbon (DOC) are of fundamental ecological and biogeochemical importance. Parallel factor (PARAFAC) analysis of excitation–emission fluorescence spectra is a common tool to characterize DOC, yet its ability to predict bacterial production (BP), bacterial respiration (BR), and bacterial growth efficiency (BGE) vary widely, potentially because inorganic nutrient limitation decouples microbial processes from their dependence on DOC composition. We used 28-d bioassays with water from 19 lakes, streams, and rivers in northern Sweden to test how much the links between bacterial metabolism and fluorescence PARAFAC components depend on experimental additions of inorganic nutrients. We found a significant interaction effect between nutrient addition and fluorescence on carbon-specific BP, and weak evidence for influence on BGE by the same interaction (p = 0.1), but no corresponding interaction effect on BR. A practical implication of this interaction was that fluorescence components could explain more than twice as much of the variability in carbon-specific BP (R2 = 0.90) and BGE (R2 = 0.70) after nitrogen and phosphorus addition, compared with control incubations. Our results suggest that an increased supply of labile DOC relative to ambient phosphorus and nitrogen induces gradually larger degrees of nutrient limitation of BP, which in turn decouple BP and BGE from fluorescence signals. Thus, while fluorescence does contain precise information about the degree to which DOC can support microbial processes, this information may be hidden in field studies due to nutrient limitation of bacterial metabolism.