In order to develop subsurface CO2 storage as a viable engineered mechanism to reduce the emission of CO2 into the atmosphere, any potential leakage of injected supercritical CO2 (SC-CO2) from the deep subsurface to the atmosphere must be reduced. Here, we investigate the utility of biofilms, which are microorganism assemblages firmly attached to a surface, as a means of reducing the permeability of deep subsurface porous geological matrices under high pressure and in the presence of SC-CO2, using a unique high pressure (8.9 MPa), moderate temperature (32 °C) flow reactor containing 40 millidarcy Berea sandstone cores. The flow reactor containing the sandstone core was inoculated with the biofilm forming organism Shewanella fridgidimarina. Electron microscopy of the rock core revealed substantial biofilm growth and accumulation under high-pressure conditions in the rock pore space which caused >95% reduction in core permeability. Permeability increased only slightly in response to SC-CO2 challenges of up to 71 h and starvation for up to 363 h in length. Viable population assays of microorganisms in the effluent indicated survival of the cells following SC-CO2 challenges and starvation, although S. fridgidimarina was succeeded by Bacillus mojavensis and Citrobacter sp. which were native in the core. These observations suggest that engineered biofilm barriers may be used to enhance the geologic sequestration of atmospheric CO2.