Bacterial whole-cell bioreporters are practical and reliable analytical tools to assess the toxicity and bioavailability of environmental contaminants, yet evidence has shown that their performance could be affected by different carbon sources. This paper evaluated the influence of carbon sources on the recA gene (ACIAD1385) in a DNA damage-inducible recA::luxCDABE Acinetobacter bioreporter and optimized the induction conditions for its practical application in environmental monitoring. Different carbon sources, including LB, potassium acetate (MMA), sodium citrate (MMC), sodium pyruvate (MMP), and sodium succinate (MMS), significantly influenced (p < 0.05) the bioluminescence intensity of the genotoxicity bioreporter. A reverse transcription quantitative PCR (RT-qPCR) showed the different expression levels of the DNA damage-inducible gene recA (p < 0.05), suggesting that carbon sources influenced the DNA damage response in the Acinetobacter bioreporter at the transcriptional level. Additionally, proteomic analysis identified 122 proteins that were differentially expressed after exposure to mitomycin C in defined media and LB, and 5 of them were related to the DNA damage response, indicating the effects of carbon sources on the DNA damage response in Acinetobacter at the translational level. The repression effect caused by the rich medium, LB, was possibly related to the mechanism of carbon catabolite repression. Our results suggest that the practical application of Acinetobacter bioreporters to the genotoxicity assessment of polycyclic aromatic hydrocarbon (PAH)-contaminated soils could be significantly improved by using a standard medium of defined composition, as this could increase their sensitivity.