Oil spillages lead to the formation of hydrocarbon and metal mixtures possessing effects on alkane-degrading bacteria that are responsible for the bioremediation of oil-contaminated soils and waters. Studies of bacterial responses to the mixture of petroleum and metal can inform appropriate strategies for bioremediation. We employed a luminescent bioreporter Acinetobacter baylyi ADPWH_recA with alkane degradation capability to evaluate the combined effects from heavy metals (Cd, Pb and Cu) and alkanes (dodecane, tetradecane, hexadecane and octadecane). Bioluminescent ratios of ADPWH_recA in single Cd or Pb treatments ranged from 0.25 to 1.98, indicating both genotoxicity and cytotoxicity of these two metals, while ratios <1.0 postexposure to Cu showed its cytotoxic impacts on ADPWH_recA bioreporter. Metal mixtures exhibited enhanced antagonistic effects (Ti > 4.0) determined by the Toxic Unit model. With 100 mg/L alkane, the morbidity of ADPWH-recA reduced to <20%, showing the inhibition of alkanes on Cd toxicity. Exposed to the metal mixture containing 10 mg/L Cu, the weak binding affinity of Cu with alkanes contributed to a high morbidity of > 85% in ADPWH_recA cells. This study provides a new way to understand the toxicity of mixture contaminants, which can help to optimize treatment efficiencies of bacterial remediation for oil contamination.