Dryland rivers make up a significant portion of the Australia’s inland freshwater networks, comprising almost the entirety of the Murray-Darling Basin. During periods of no flow, river networks in these areas contract to form isolated “waterholes” that are critically important for aquatic biota within. In periods of no flow, Australia’s dryland rivers display traits both lentic (e.g., stratification) and lotic (e.g., benthic productivity), and life within these waterholes is critically dependent on basal metabolic processes that remain poorly understood in relation to the highly variable flows. We investigated the ecohydrology of 4 waterholes in the Northern Murray Darling Basin to determine how hydrology, including cease-to-flow periods, impacts stream metabolism. Using gross primary productivity (GPP), ecosystem respiration (ER), and trophic structure as our metabolic measures, we sought to identify the hydrologic and environmental drivers of metabolic structure and function at multiple scales. We found that while suspended fine sediment limited productivity, as well as other biophysical parameters, across all phases of the hydrograph, the metabolic responses of dryland rivers to periods of flow and no flow were significantly different. At a catchment scale, the upstream drainage area was the most influential factor determining average daily metabolic rates, but no catchment factors adequately explained trophic state. At the reach scale, storm and drought frequency affected GPP differently, while higher seasonal flow volume generally indicated lower rates of GPP and ER, as well as greater heterotrophy. Contrary to expectation, ER varied inversely with GPP during surface runoff events. Understanding the structure and function of stream ecology in all hydrologic phases, as well as factors influencing metabolism, is important for managing native aquatic life that depends on the water in these systems. This study offers a framework for identifying key hydrologic and environmental controls to stream metabolism and the basic resources that sustain life in a highly variable environment.