Chromate can be considered a potent environmental contaminant and consequently, an understanding of chromate availability and toxicity to soil biology is essential for effective ecological assessment of metal impact in soils. This study shows the response of two bacterial bioreporters, pCHRGFP1 Escherichia coli and pCHRGFP2 Ochrobactrum tritici, to increasing concentrations of chromate in two different soils. The bioreporters, carrying the regulatory gene chrB transcriptionally fused to the gfp reporter system, exhibited different features. In both, the fluorescence signal and the chromate concentration could be linearly correlated but E. coli biosensor functioned within the range of 0.5–2μM and O. tritici biosensor within 2–10μM chromate. The bioreporters were validated through comparative measurements using the chemical chromate methods of diphenylcarbazide and ionic chromatography. The bacterial sensors were used for the estimation of bioavailable fraction of chromate in a natural soil and OECD artificial soil, both spiked with chromate in increasing concentrations of 0–120mgCr(VI)kg−1 of soil. OECD soil showed a faster chromate decrease comparing to the natural soil. The toxicity of soils amended with chromate was also evaluated by ecotoxicological tests through collembolan reproduction tests using Folsomia candida as test organism. Significant correlations were found between collembolans reproduction and chromate concentration in soil (lower at high chromate concentrations) measured by biosensors. Data obtained showed that the biosensors tested are sensitive to chromate presence in soil and may constitute a rapid and efficient method to measure chromate availability in soils.