We report on a novel approach to predict the mode of genotoxic action of chemicals using a series of DNA damage specific bioluminescent bacteria. For this, a group of seven different DNA damage sensing recombinant bioluminescent strains were employed. Each of these strains was tested against model DNA damaging agents, such as mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), nalidixic acid (Nal) and 4-nitroquinoline N-oxide (4-NQO). These biosensors were grouped based on their responses to a specific mode of genotoxic action, such as (a) DNA damage cascade response (biosensor with nrdA-, dinI- and sbmC-lux), (b) SOS response or DNA repair (strains carrying recA-, recN- and sulA-lux), and (c) DNA damage potentially by alkylation (biosensor with alkA-lux). The differential response patterns and its strength of these strains to various model genotoxicants allowed classifying the chemical's potential genotoxic mode. Therefore, it is possible to elucidate and classify the mode of genotoxic impacts of an unknown sample and that together they may be utilized in the pre-screening steps of new drugs, newly synthesized chemicals, food and environmental contaminants.