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Emergence of life-threatening fungal infections like systemic candidiasis concurrently with bacterial infections and limitations of current antifungal therapies warrants the discovery of novel inhibitors. We identified besifloxacin (BS), an FDA-approved antibacterial, as a potent antifungal inhibitor. A combination of besifloxacin with fluconazole showed a positive synergy (δ = 29.58) resulting in 80% inhibition of microbial growth. BS was able to reduce the MIC of FLC from 2mg/L to 0.5 mg/L when used in combination. Additionally in murine systemic Candida infection, BS reduced fungal load by 83% in mice kidneys at a dose of 100 mg/kg/day. The findings demonstrated the antifungal potential of BS, proposing its use in combination therapy with fluconazole to combat resistance through alternative mechanisms.