Recent confirmation of widespread microbial activity in the deep subsurface has raised the question whether microbes were transported to their current residence from the surface or whether they have survived in situ since sediment deposition. As a part of a larger study addressing these and related questions, we have characterized the microbiology and hydrogeology of a Late Cretaceous sandstone and shale sequence in the southeastern San Juan Basin, New Mexico, near a 3.39 Ma volcanic intrusion. Deep core samples were analyzed for microbial activity to assess recolonization of the previously sterilized zone around the intrusion. Groundwater geochemistry and isotopic data were used to improve the understanding of the flow regime. We modeled the geochemical evolution of the groundwater from the recharge area to each sample location and used the resultant mass transfers to correct measured 14C activities. The 14C ages provided the basis for calibrating a cross-sectional flow model that intersects the intrusion. Based on microbial activity data, hydrogeologic modeling results supported the inference that groundwater velocities were adequate to transport microbes into the previously sterilized region in the time since the volcanic intrusion. Evidence of upward groundwater flow near the intrusion and high vertical hydraulic conductivities for shale suggest considerable hydraulic connection between lithologic units, which may influence the nutrient distribution and promote enhanced microbial activity near lithologic interfaces.