This study evaluated the in vitro anti-anaerobic activity and spectrum of levornidazole, its metabolites and comparators against 375 clinical isolates of anaerobic bacteria, including Gram-negative bacilli (181 strains), Gram-negative cocci (11 strains), Gram-positive bacilli (139 strains) and Gram-positive cocci (44 strains), covering 34 species. Minimum inhibitory concentrations (MICs) of levornidazole, its five metabolites and three comparators against these anaerobic isolates were determined by the agar dilution method. Minimum bactericidal concentrations (MBCs) of levornidazole and metronidazole were measured against 22 strains of Bacteroides fragilis. Levornidazole showed good activity against B. fragilis, other Bacteroides spp., Clostridium difficile, Clostridium perfringens and Peptostreptococcus magnus, evidenced by MIC90 values of 0.5, 1, 0.25, 2 and 1mg/L, respectively. The activity of levornidazole and the comparators was poor for Veillonella spp. Generally, levornidazole displayed activity similar to or slightly higher than that of metronidazole, ornidazole and dextrornidazole against anaerobic Gram-negative bacilli, Gram-positive bacilli and Gram-positive cocci, especially B. fragilis. Favourable anti-anaerobic activity was also seen with levornidazole metabolites M1 and M4 but not M2, M3 or M5. For the 22 clinical B. fragilis strains, MBC50 and MBC90 values of levornidazole were 2mg/L and 4mg/L, respectively. Both MBC50/MIC50 and MBC90/MIC90 ratios of levornidazole were 4, similar to those of metronidazole. Levornidazole is an important anti-anaerobic option in clinical settings in terms of its potent and broad-spectrum in vitro activity, bactericidal property, and the anti-anaerobic activity of its metabolites M1 and M4.