The environmental fate and potency of mutagenic compounds is of growing concern. This has necessitated the development and application of rapid assays to screen large numbers of samples for their genotoxic and carcinogenic effects. Despite the development of biosensors for genotoxicity assessment, these have not been calibrated against traditional microbial bioassays. In this study, assays using the SOS-lux-marked microbial biosensors Escherichia coli K12C600 and E. coli DPD1718 were refined and optimised to screen selected mutagenic chemicals. The response of the biosensors was compared with the mutagenic response of the traditional Salmonella mutagenicity assay. For the chemicals tested (acridine, B[a]A, B[a]P, chrysene, mitomycin C and sodium azide), E. coli DPD1718 was consistently more sensitive than E. coli K12C600. The biosensors were of comparable sensitivity to the Salmonella assay but were more rapid, reproducible and easier to measure. These data validate the adoption of optimised assays making use of microbial biosensors for routine screening of test chemicals.