Microbial Cr(VI) reduction in groundwater aquifer media was investigated in microcosm reactors extracted from Cr(VI) contaminated sites in South Africa. The reactors were operated under an influent Cr(VI) concentration of 40 mg/L to simulate the current Cr(VI) level at the contaminated site. Near complete Cr(VI) removal was observed in microcosm reactors inoculated with Cr(VI) reducing bacteria from dried activated sludge collected from a treatment plant receiving periodic loadings of Cr(VI). The best performance was observed under low hydraulic loading (flow rate, Q=0.310 cm3/hr). Microbial culture characterisation results showed a change in culture composition after 17 days of reactor operation, indicating Bacillus and Lysinibacillus species as the most dominant organisms in reactors that reduced Cr(VI). The predominance of Bacillus and Lysinibacillus species was either due to resilience against toxicity or adaptation to the changing conditions in the reactor. This research was the initial step towards the development of an in situ bioremediation process to contain the spread of a Cr(VI) plume in a groundwater aquifer at contaminated site in Brits, South Africa. South Africa holds about 72% percent of the world’s chromium resources, the majority of which is mined in the North Eastern region of the country formally known as Transvaal.