The in vitro activity of gatifloxacin, a new fluoroquinolone, was compared to that of five other fluoroquinolones against 105 Stenotrophomonas maltophilia isolates and 52 Burkholderia spp. isolates. The gatifloxacin MICs were determined using the broth microdilution method and the E test (AB Biodisk, Sweden); these methods were compared for test accuracy, and 5 μg disk zone diameters were compared for interpretive accuracy using the standardized disk diffusion method. In terms of potency, gatifloxacin was most similar to sparfloxacin and trovafloxacin against Stenotrophomonas maltophilia (MIC50, 0.5–1 mg/l) and Burkholderia spp. (MIC50, 1–2 mg/l). This activity was greater than that of ciprofloxacin, levofloxacin or ofloxacin (MIC50, ≥2 mg/l) against Stenotrophomonas maltophilia isolates but comparable to that of levofloxacin against the Burkholderia spp. (60% susceptible at ≤2 mg/l). The E test results compared well with the reference dilution test results (81–97% at ±1 log2 dilution). The disk diffusion test using previously suggested breakpoints for other bacteria (≥18 mm or ≤2 mg/l for susceptible and ≤14 mm or ≥8 mg/l for resistant) also performed well, at >90% categorical agreement. The activity of gatifloxacin is comparable to that of other newer quinolones against isolates of Stenotrophomonas maltophilia and Burkholderia spp., and susceptibility testing using simple qualitative and quantitative methods appears to function well with these drug/organism combinations.