Abstract The objective of this study was to evaluate the in-vitro activity of levofloxacin, ofloxacin and d -ofloxacin compared with ciprofloxacin, norfloxacin and sparfloxacin against coryneform bacteria and Listeria monocytogenes isolated from clinical samples. The following organisms (and number of strains) were studied: Corynebacterium jeikeium (20), Corynebacterium urealyticum (20), Corynebacterium minutissimum (20), Corynebacterium striatum (20), Corynebacterium amycolatum (30), Brevibacterium spp. (15) and Listeria monocytogenes (15). Antimicrobial activity was determined by microdilution using cation-adjusted Mueller–Hinton broth supplemented with 0.5% Tween 80 when testing C. jeikeium or C. urealyticum. Fluoroquinolones were used in the range 0.015–16 mg/L. Plates were incubated in air at 35°C for 18–20 h (24 h when testing C. jeikeium or C. urealyticum ). The following MIC 50 values were obtained for all 140 organisms tested: levofloxacin, 1 mg/L; ofloxacin, 2 mg/L; d -ofloxacin, >16 mg/L; ciprofloxacin, 1 mg/L; norfloxacin, 16 mg/L; sparfloxacin, 1 mg/L. MIC 90 values were >16 mg/L for all test antibiotics with the exception of levofloxacin, which had an MIC 90 value of 16 mg/L. At a concentration of 2 mg/L, levofloxacin inhibited all L. monocytogenes strains and 35–93% of the remaining species. MIC 90 values of ofloxacin were one dilution step higher than those of levofloxacin against C. minutissimum,C. striatum,Brevibacterium spp. and L. monocytogenes. Levofloxacin showed similar ( C. jeikeium,C. urealyticum,C. amycolatum and Brevibacterium spp.) or greater ( C. minutissimum and C. striatum ) activity than ciprofloxacin and sparfloxacin, and higher than d -ofloxacin or norfloxacin against all species studied. In conclusion, levofloxacin was the most active of the six fluoroquinolones evaluated against coryneform bacteria isolated from clinical samples and could therefore be a promising treatment option in this setting.