Stenotrophomonas maltophilia is an opportunistic pathogen that causes respiratory and urinary tract infections, as well as wound infections in immunocompromised patients. This pathogen is difficult to treat due to increased resistance to many antimicrobial agents. We investigated the in vitro biofilm formation of S. maltophilia, including effects of fluoroquinolones (FQs) and azithromycin on biofilm formation. The organism initiated attachment to polystyrene surfaces after a 4 h incubation period, and reached maximal growth at 18-24 h. In the presence of FQs (moxifloxacin, levofloxacin or ciprofloxacin), the biofilm biomass was significantly reduced (P < 0.05). A lower concentration of moxifloxacin (10 μg/mL) exhibited a better inhibiting effect on biofilm formation than 100 μg/mL (P < 0.01), but with no difference in effect compared to the 50 μg/mL concentration (P > 0.05). However, the inhibitory effects of 10 μg/mL of levofloxacin or ciprofloxacin were slightly less pronounced than those of the higher concentrations. A combination of azithromycin and FQs significantly reduced the biofilm inhibiting effect on S. maltophilia preformed biofilms compared to azithromycin or FQs alone. We conclude that early use of clinically acceptable concentrations of FQs, especially moxifloxacin (10 μg/mL), may possibly inhibit biofilm formation by S. maltophilia. Our study provides an experimental basis for a possible optimal treatment strategy for S. maltophilia biofilm-related infections.