Antibacterial properties of 3′,4′-difluoroquercetin (di-F-Q), a fluorine-substituted stable quercetin derivative, were investigated. Even though di-F-Q itself did not show interesting antibacterial activity, treatment of the Staphylococcus aureus strains with di-F-Q resulted in a dose-dependent reduction in biofilm formation with IC 50 values of 1.8 ~ 5.3 mg/L. Also, the antibacterial activity of ceftazidime (CAZ) against carbapenem-resistant Pseudomonas aeruginosa (CRPA) showed eightfold decrease upon combination with di-F-Q. Assessment of the antimicrobial activity of CAZ in combination with di-F-Q against 50 clinical isolates of P. aeruginosa confirmed 15.7% increase in the percentages of susceptible P. aeruginosa isolates upon addition of di-F-Q to CAZ. Further mechanistic studies revealed that di-F-Q affected the antibiotics efflux system in CRPA but not the β-lactamase activity. Thus, di-F-Q was almost equally effective as carbonyl cyanide m-chlorophenyl hydrazine in inhibiting antibiotic efflux by P. aeruginosa. In vivo evaluation of the therapeutic efficacy of CAZ-(di-F-Q) combination against P. aeruginosa showed 20% of the mice treated with CAZ-(di-F-Q) survived after 7 days in IMP carbapenemase-producing multidrug-resistant P. aeruginosa infection group while no mice treated with CAZ alone survived after 2 days. Taken together, di-F-Q demonstrated unique strain-specific antimicrobial properties including anti-biofilm and antibiotic-potentiating activity against S. aureus and P. aeruginosa, respectively. Quercetin (Fig. 1) is one of the most famous flavonoids, and it is characterized by broad spectrum biological activity including antioxidant, anti-inflammatory, antiviral, and anticancer effects 1. Quercetin has also been reported to have a potential as an antibacterial agent 1 , and its anti-biofilm activity against clinical isolates of gram-positive bacteria, albeit low 2 , is noteworthy. However, several unfavorable physicochemical properties of quercetin such as low chemical stability limits its pharmaceutical use. Previously, in order to tackle this problem, we prepared 3′,4′-difluoroquercetin (di-F-Q, Fig. 1) through bioisosteric replacement of the catecholic hydroxyl groups of quercetin with fluorine atoms, which showed significantly improved chemical stability as well as anticancer activity compared with quercetin 3. In this context, antibacterial properties of di-F-Q appears worth investigating and, in this study, we attempted to evaluate antibacterial, anti-biofilm, and antibiotic-potentiating activity of di-F-Q. Materials and methods Materials. Dimethyl sulfoxide (DMSO), crystal violet and phosphate buffered saline (PBS) were purchased from Merck (St. Louis, MO, USA). Mueller-Hinton broth (MHB) and tryptic soy broth (TSB) were obtained from BD Biosciences (San Jose, CA, USA) and Fisher Scientific (Pittsburgh, PA, USA), respectively.