Groundwater microbial community dynamics are poorly understood due to the challenges associated with accessing subsurface environments. In particular, microbial interactions and their impact on the subsurface carbon cycle remain unclear. In the present project, stable isotope probing with uniformly labeled [(13)C]-acetate was used to identify metabolically active and inactive bacterial populations based on their ability to assimilate acetate and/or its metabolites. Furthermore, we assessed whether substrate availability (bottom-up control) or grazing mortality (top-down control) played a greater role in shaping bacterial community composition by separately manipulating the organic carbon supply and the protozoan grazer population. A community fingerprinting technique, terminal restriction fragment length polymorphism, revealed that the bacterial community was not affected by changes in acetate availability but was significantly altered by the removal of protozoan grazers. In silico identification of terminal restriction fragments and 16S rRNA gene sequences from clone libraries revealed a bacterial community dominated by Proteobacteria, Firmicutes, and Bacteroidetes. Elucidation of the factors that structure the bacterial community will improve our understanding of the bacterial role in the carbon cycle of this important subterranean environment.