Benzydamine is an anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to benzydamine N-oxide; however, benzydamine N-demethylation is also catalyzed by liver microsomes. In this study, benzydamine N-oxygenation and N-demethylation mediated by liver microsomes from rats, dogs, monkeys, and humans were characterized comprehensively. Values of the maximum velocity/Michaelis constant ratio for benzydamine N-oxygenation by liver microsomes from dogs and rats were higher than those from monkeys and humans, despite roughly similar rates of N-demethylation in the four species. Benzydamine N-oxygenation by liver microsomes was extensively suppressed by preheating liver microsomes at 45 °C for 5 min or at 37 °C for 5–10 min without NADPH, and benzydamine N-demethylation was strongly inhibited by 1-aminbobenztriazole. Liver microsomal benzydamine N-oxygenation was inhibited by dimethyl sulfoxide and methimazole, whereas N-demethylation was inhibited by quinidine. High benzydamine N-oxygenation activities of recombinant human FMO1 and FMO3 and human kidney microsomes were observed at pH 8.4, whereas N-demethylation by cytochrome P450 2D6 was faster at pH 7.4. These results suggest that benzydamine N-oxygenation and N-demethylation are mediated by FMO1/3 and P450s, respectively, and that the contribution of FMO to metabolic eliminations of new drug candidates might be underestimated under certain experimental conditions suitable for P450 enzymes.