River regulation by dams and reservoirs can alter flow regime and water chemistry, impacting ecological functioning of downstream ecosystems. Unregulated tributaries can restore certain water quality parameters (e.g., water temperature, sediment) of a regulated mainstem, but less is known about dissolved organic matter (DOM) processing and transport despite its ecological importance. This study examines the water quality, especially DOM concentration and composition of a regulated and unregulated river under different flow conditions in south-east Australia. For this, we collected water samples from three sites: (i) regulated (downstream from reservoir and upstream from confluence), (ii) tributary and (iii) confluence (downstream from where the tributary joins the regulated river) to monitor DOM (dissolved organic carbon (DOC), organic nitrogen (DON) and organic phosphorus (DOP)) concentrations and composition using fluorescence spectroscopy and size exclusion chromatography. The tributary was significantly different from regulated and confluence sites during base flow conditions and contained higher DOM concentrations (DOC, DON and DOP), consisting of more aromatic, high molecular weight, terrestrially derived DOM constituents. In contrast, storm pulses of tributary increased DOM concentrations in the confluence and altered the DOM composition to resemble the tributary site more greatly than the regulated site. The supply of these different kinds of DOM from tributaries to regulated rivers may enhance primary productivity and ecological functioning of downstream ecosystems.