The metabolism of dissolved organic carbon (DOC) along fluvial networks determines what fraction of organic matter is exported to the ocean. Although it is thought fresh rather than older DOC is preferred by bacteria, old DOC can also be highly bioavailable to stream bacterial communities. In strongly seasonal and oligotrophic regions, we argue that groundwater inputs of old DOC may increase the bioavailability of stream organic matter. We sampled 22 streams along a gradient of size (wetted widths from 1 to 60 m) and one groundwater spring in the Kimberley region of northwest Australia to determine how the age and bioavailability of streamwater DOC varied with stream size. Our hypothesis was that stream DOC would become more enriched in 14C (younger) and less bioavailable as streams increased in size and depleted 14C-DOC was metabolized by stream microbial communities. We also used fluorescence characterization of DOC, ultraviolet absorbance at 254nm (SUVA254), δ13C-DOC and lignin phenol yields to assess how these indicators of DOC character influenced the bioavailability and age of stream DOC. Stream evaporation/inflow ratios (E/I, used as a proxy for catchment water residence time), determined from changes in stream δ18O along the gradient of stream size, were positively related to DOC concentration and carbon-normalized lignin yields, while δ13C-DOC became more depleted with increasing E/I. Stream Δ14C-DOC varied from −452.1‰ (groundwater) to 48.9‰ and showed progressive enrichment as streams increased in size and accumulated DOC mainly from terrestrial plant material. Older DOC corresponded to higher bioavailability (R2 = 0.67, P < 0.01), suggesting that old bioavailable DOC, which has escaped from subterranean food webs utilizing 14C-depleted carbon, is common to one of the oldest landscapes on earth. Therefore, rapid biotic uptake of old bioavailable DOC originating in groundwater springs and the accumulation of modern, terrestrially derived DOC work in opposite directions affecting DOC dynamics along fluvial networks. We suggest the metabolism of old DOC along fluvial networks provides a biogeochemical link between non-contemporary carbon fixation and modern river productivity.