The main goal of this research was to evaluate whether the mixture of fresh labile dissolved organic matter (DOM) and accumulated refractory DOM influences bacterial production, respiration, and growth efficiency (BGE) in aquatic ecosystems. Bacterial batch cultures were set up using DOM leached from aquatic macrophytes as the fresh DOM pool and DOM accumulated from a tropical humic lagoon. Two sets of experiments were performed and bacterial growth was followed in cultures composed of each carbon substrate (first experiment) and by carbon substrates combined (second experiment), with and without the addition of nitrogen and phosphorus. In both experiments, bacterial production, respiration, and BGE were always higher in cultures with N and P additions, indicating a consistent inorganic nutrient limitation. Bacterial production, respiration, and BGE were higher in cultures set up with leachate DOM than in cultures set up with humic DOM, indicating that the quality of the organic matter pool influenced the bacterial growth. Bacterial production and respiration were higher in the mixture of substrates (second experiment) than expected by bacterial production and respiration in single substrate cultures (first experiment). We suggest that the differences in the concentration of some compounds between DOM sources, the co-metabolism on carbon compound decomposition, and the higher diversity of molecules possibly support a greater bacterial diversity which might explain the higher bacterial growth observed. Finally, our results indicate that the mixture of fresh labile and accumulated refractory DOM that naturally occurs in aquatic ecosystems could accelerate the bacterial growth and bacterial DOM removal.