Background <p><i>Stenotrophomonas maltophilia</i> (<i>S. maltophilia</i>) drug resistance is a crisis worldwide. Our objective was evaluation of synergistic antibacterial and anti-biofilm effects of fluoxetine and quercetin against carbapenemase producing <i>S. maltophilia</i> (CR-St) clinical isolates.</p> Methods <p>Thirty CR-St isolates were recovered from various clinical specimens. Various concentrations of imipenem, quercetin and fluoxetine were prepared for the antibacterial tests. The expression of SmeE efflux pump gene was evaluated in exposure to quercetin and fluoxetine using quantitative real-time PCR. Anti-biofilm effects and time kill assay tests were also performed.</p> Results <p>The isolates exhibited high level resistance to imipenem (MIC: 8–64&#xa0;µg/mL) but inferred resistance to fluoxetine (MIC: 128–256&#xa0;µg/mL) and quercetin (MIC: 128–512&#xa0;µg/mL) at higher concentrations. The fluoxetine and quercetin (1:1 ratio) had strong antibacterial activity since the synergistic effects of the two compounds significantly reduced the MIC values and also provided bactericidal activity data in time-kill studies. All isolates produced biofilms, and most produced moderate to strong biofilms. Fluoxetine and quercetin inhibited biofilm formation and disrupted pre-formed biofilms to lesser degree. The fluoxetine and quercetin combination had synergistic capabilities by decreasing biofilm formation up to 85% and disruption of mature biofilms with some observed structural destruction. The expression of SmeE efflux pump gene was decreased in exposure to quercetin by 2.2 fold (<i>p</i> &lt; 0.0001).</p> Conclusion <p>Quercetin and fluoxetine could potentially exert anti-biofilm and antibacterial effects against CR-St with synergistic properties in vitro.</p>