Biological remediation of Cr(VI) contaminated soil and groundwater is an emerging field. In this study, the in situ bioremediation technology for treating Cr(VI) contaminated groundwater aquifers was evaluated using a laboratory microcosm system. The study was conducted using columns with five equally spaced intermediate sampling ports along the length to facilitate finite difference modelling of the Cr(VI) concentration profile within the column. Cr(VI) concentration was continuously measured in the influent, in five equally spaced intermediate ports within the column and in the effluent port. The change or the shift in microbial community within the inoculated column was also monitored due to exposure to toxic conditions after seven weeks of operation using the 16S rRNA genotype fingerprinting method. The effect of introducing a natural carbon source (sawdust) in inoculated columns in comparison with the performance of sterile controls under various loading conditions was also evaluated. Near complete Cr(VI) removal was achieved in an inoculated carbon source reactor, whereas only 69.5% of Cr(VI) removal was achieved in an inoculated column without an added carbon source after 4 days of operation at 20 mg/L. In a sterile control reactor less than 2% of Cr(VI) was removed after 4 days of operation at 20 mg/L. Experimental cores demonstrated a successful Cr(VI) reduction process in the simulated microbial barrier system that was evaluated internally. The model that simulates Cr(VI) removal and transport in the subsoil environment was developed. The Cr(VI) mass balance model across the reactor that accounts for the flow characteristics and biological removal mechanism successfully captured the trends of Cr(VI) response profiles under quasi-steady state conditions for different loading conditions. This study demonstrate the potential of applying effective Cr(VI) reducers in the reactive barrier systems to contain or attenuate the spread of Cr(VI) contaminant in groundwater aquifer systems. The finite difference model developed in this study to evaluate the behaviour of Cr(VI) in the reactor could contribute towards improved designs of future in situ bioremediation systems that can be implemented for remediation of Cr(VI) on site.