Pathogenic bacteria experience diverse stresses induced by host cells during infection and have developed intricate systems to trigger appropriate responses. Bacterial stress responses have been reported to defend against these stresses and cross-protect bacteria from antibiotic attack. In this study, we aimed to assess whether oxidative stress affects bacterial susceptibility to fluoroquinolone (FQ) and the underlying mechanism. Stenotrophomonas maltophilia, a species with high genetic diversity, is distributed ubiquitously and is an emerging multidrug-resistant opportunistic pathogen. FQs are among the limited antibiotic treatment options for S. maltophilia infection. The minimum inhibitory concentrations (MICs) of 103 S. maltophilia clinical isolates against ciprofloxacin (CIP) and levofloxacin (LVX) were determined using the agar dilution method in Mueller-Hinton plates with or without menadione (MD), a superoxide generator. The resistance rates for ciprofloxacin and levofloxacin were 40% and 18% in the MD-null group and increased to 91% and 23%, respectively, in the MD-treated group. Of the 103 isolates tested, 54% and 27% had elevated MICs against ciprofloxacin and levofloxacin, respectively, in the presence of MD. The involvement of oxidative stress responses in the MD-mediated FQ resistance was further assessed by mutants construction and viability assay. Among the 16 oxidative stress alleviation systems evaluated,