Dissolved organic matter (DOM) within freshwaters is essential for broad ecosystem function. The concentration and type of DOM within rivers depends on the relative contributions of allochthonous sources and the production and consumption of DOM by microbes. In this work we have examined the temporal patterns in DOM quality in three lowland rivers in dryland Australia using fluorescence excitation emission scans. We assessed the production and consumption of DOM within light and dark bottle assays to quantify the relative contribution of bacteria and algae to the DOM pool and simultaneously assessed whether the systems were autotrophic or heterotrophic. DOM varied temporally within the three river systems over the course of the study period. Characterisation of DOM within light and dark bottles following a 6-hour incubation revealed microbial consumption of a humic-like component and production of protein-like components similar in nature to the amino acids tryptophan and tyrosine. The lack of a significant difference in DOM quality between the light and dark bottles indicated that the protein-like DOM is likely derived from bacterial activity. Respiration was shown to be higher than gross primary production in both whole river and bottle assays, yielding negative net production values and demonstrating that these rivers were predominately heterotrophic. Our work suggests that bacterial metabolism of DOM may be a significant contributor to the production of protein-like components within heterotrophic freshwater systems.