BACKGROUND: Aeromonas infections pose a significant threat associated with high mortality rates. This study investigates the potential of mitomycin C (MMC), an anticancer drug, as a novel antimicrobial agent against Aeromonas infections. METHODS: We evaluated the minimum inhibitory concentrations (MICs) of MMC and antibiotics against clinical Aeromonas isolates using broth microdilution. Synergistic effects of MMC with antibiotics were determined via time-kill studies. MMC's intracellular killing effects were analyzed using a representative Aeromonas isolate. Efficacy of combined therapies was assessed in a neutropenic mouse model. MMC-induced SOS response was evaluated using cell elongation method, and RNA extraction and quantitative real-time PCR. RESULTS: Combining 1/8⨯ MIC of mitomycin C (MMC) with either 1⨯ or 1/2⨯ MIC of LVX demonstrated significant synergistic effects over 24 h in vitro. In a neutropenic mouse model, the combination of MMC (2 mg/kg or 1 mg/kg) with LVX achieved survival rates of 100 % and 80 %, respectively, compared to 0 % survival with monotherapy. MMC induced marked cell elongation and division inhibition in response to escalating doses. However, the combination therapy's enhancement did not surpass the effects of individual drug treatments. Notably, combination therapy reduced recA activator levels below those observed with either drug alone, suggesting rapid bacterial cell death curtailed further expression of recA and lexA. Alternatively, extensive DNA damage may have overwhelmed bacterial DNA repair mechanisms, rendering them ineffective. CONCLUSIONS: These findings suggest that MMC may serve as a potential antimicrobial agent, particularly when used in combination with antibiotics. The integration of MMC with antibiotic therapy offers a promising approach for the treatment of Aeromonas infections and holds potential for future clinical applications.