Studies exploring how different sources of dissolved organic matter (DOM) influence in-stream dissolved organic carbon (DOC) uptake at the ecosystem scale are scarce in the literature. To fill this knowledge gap, we examined the relationship between DOM sources and in-stream net DOC uptake (UDOC) in a sub-humid Mediterranean stream. We considered four reach-scale scenarios occurring under natural conditions that differed in predominant DOM sources (groundwater, leaf litter, and/or upstream water). Results showed that groundwater inputs favored in-stream net DOC uptake, while leaf litter inputs promoted in-stream net DOC release. However, there was no clear effect of DOM source mixing on the magnitude of UDOC. Further, the variability in UDOC within and among scenarios was mostly explained by stream DOC concentration, suggesting that DOC availability limits microbial activity in this stream. DOM composition became a controlling factor of UDOC variability only during the leaf litter period, when stream DOC concentration was the highest. Together, these results suggest that the capacity of headwater forested streams to process DOC is closely tied to the availability of different DOM sources and how they vary over time and along the river network.