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The effect of Cr⁶⁺-contaminated groundwater was assessed using thiosulfate-oxidizing bacteria (TOB). Electrical conductivity (EC), pH, and sulfate production were determined based on thiosulfate oxidation. Final pH values in the different test treatments of Cr⁶⁺-contaminated groundwater (50–1000 μg Cr⁶⁺ L⁻¹) ranged from 2.02 ± 0.09 to 7.76 ± 0.07 and EC ranged from 5.95 ± 0.03 to 3.63 ± 0.03 mS cm⁻¹. Inhibition of TOB due to Cr⁶⁺ was between 16.7% and 100%, with higher levels of inhibition occurring at higher Cr⁶⁺ concentrations. The median effective concentration (EC₅₀) was 78.96 μg Cr⁶⁺ L⁻¹. These data demonstrate that TOB can detect less than 100 μg L⁻¹ of Cr⁶⁺ in the groundwater and can be used as an effective bioassay for toxicity assessment.