Headwater streams are tightly connected with the terrestrial milieu from where they receive deliveries of organic matter often through the hyporheic zone, the transition between groundwater and streamwater. Dissolved organic matter (DOM) from terrestrial sources (that is, allochthonous) enters the hyporheic zone, where it may mix with DOM from in situ production (that is, autochthonous) and where most of the microbial activity takes place. Allochthonous DOM is typically considered resistant to the microbial metabolism compared to autochthonous DOM. The composition and functioning of microbial biofilm communities in the hyporheic zone may therefore be controlled by the relative availability of allochthonous and autochthonous DOM, which may have implications for organic matter processing in stream ecosystems. Experimenting with hyporheic biofilms exposed to model allochthonous and autochthonous DOM and using 454-pyrosequencing of the 16S rRNA (targeting the “active” community composition) and of the 16S rRNA gene (targeting the “bulk” community composition), we found that allochthonous DOM may drive shifts in community composition, whereas autochthonous DOM seems to affect community composition only transiently. Our results suggest that priority effects based on resource-driven stochasticity shape community composition in the hyporheic zone. Furthermore, measurements of extracellular enzymatic activities suggest that the additions of allochthonous and autochthonous DOM had no clear effect on the function of hyporheic biofilms, indicative of functional redundancy. Our findings unravel possible microbial mechanisms that underlie the buffering capacity of the hyporheic zone and that may confer stability to stream ecosystems.