The present study applied a nano-synergistic approach to enhance besifloxacin's potency via nano-formulating besifloxacin on gold nanoparticles (Besi-AuNPs) and adding quercetin as a natural synergistic compound. In fact, a one-pot AuNP synthesis approach was applied for the generation of Besi-AuNPs, where besifloxacin itself acted as a reducing and capping agent. Characterization of Besi-AuNPs was performed by spectrophotometry, DLS, FTIR, and electron microscopy techniques. Moreover, antibacterial assessment of pure besifloxacin, Besi-AuNPs, and their combinations with quercetin were performed on Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. UV-spectra showed a peak of AuNPs at 526 nm, and the electron microscopy-based size was estimated to be 15 ± 3 nm. The effective MIC50 concentrations of besifloxacin after loading on AuNPs were reduced by approximately 50% against the tested bacterial strains. Interestingly, adding quercetin to Besi-AuNPs further enhanced their antibacterial potency, and isobologram analysis showed synergistic potential (combination index below 1) for different quercetin and Besi-AuNP combinations. However, Besi-AuNPs and quercetin combinations were most effective against Gram-positive S. aureus in comparison to Gram-negative P. aeruginosa and E. coli. Their potent activity against S. aureus has its own clinical significance, as it is one the main causative agents of ocular infection, and besifloxacin is primarily used for treating infectious eye diseases. Thus, the outcomes of the present study could be explored further to provide better medication for eye infections caused by resistant pathogens.