ObjectivesTo investigate thein vitroactivity of antibiotic combinations againstStenotrophomonas maltophiliaisolates and their associated biofilms.MethodsThirty-twoS. maltophiliaclinical isolates with at least twenty-five different pulsotypes were tested. The antibacterial activity of various antibiotic combinations against seven randomly selected planktonic and biofilm-embeddedS. maltophiliastrains with strong biofilm formation was assessed using broth methods. Extraction of bacterial genomic DNA and PCR detection of antibiotic resistance and biofilm-related genes were also performed.ResultsThe susceptibility rates of levofloxacin (LVX), fosfomycin (FOS), tigecycline (TGC) and sulfamethoxazole-trimethoprim (SXT) against 32S. maltophiliaisolates were 56.3, 71.9, 71.9 and 90.6%, respectively. Twenty-eight isolates were detected with strong biofilm formation. Antibiotic combinations, including aztreonam-clavulanic (ATM-CLA) with LVX, ceftazidime-avibactam (CZA) with LVX and SXT with TGC, exhibited potent inhibitory activity against these isolates with strong biofilm formation. The antibiotic resistance phenotype might not be fully caused by the common antibiotic-resistance or biofilm-formation gene.ConclusionS. maltophiliaremained resistant to most antibiotics, including LVX and β-lactam/β-lactamases; however, TGC, FOS and SXT still exhibited potent activity. Although all testedS. maltophiliaisolates exhibited moderate-to-strong biofilm formation, combination therapies, especially ATM-CLA with LVX, CZA with LVX and SXT with TGC, exhibited a higher inhibitory activity for these isolates.