A whole-cell bioreporter, Acinetobacter baylyi ADPWH_recA, was used to estimate the genotoxicity and bioavailability of chromium (VI) [Cr(VI)] in contaminated soils. Upon direct exposure to pre-sonicated soil samples, ADPWH_recA gave the highest response to the genotoxicity of Cr(VI) within 5 h with a detection limit of 2 µM Cr(VI). Investigations on sites contaminated with Cr(VI) revealed that soil-associated Cr(VI) was bioavailable to the bioreporter although it could not be extracted into the aqueous phase. The physical and chemical properties of soil might influence the bioavailability of Cr(VI), and higher genotoxicity was found in soils with a lower pH. This whole cell bioreporter approach makes it feasible to evaluate the bioavailability and genotoxicity of Cr(VI)-contaminated soils to uncover their potential impact on human health.