Metronidazole (MTZ) is an antibacterial/antiprotozoal drug used to treat infections. This study aimed to assess the safety of two pharmacologically active MTZ analogs, MTZ-Ms and MTZ-I, in an attempt to provide scientific evidences that justify further investments in these drugs. The acute toxicity studies were performed using 300 or 2000 mg/kg doses. No treatment-related mortality occurred in MTZ and MTZ-Ms groups. By contrast, one death was detected when using 2000 mg/kg of MTZ-I. In the subacute toxicity studies, 200, 400, or 600 mg/kg doses of MTZ and MTZ-I, and a 1000 mg/kg dose of MTZ-Ms, were applied. No abnormal behavior was observed in the MTZ and MTZ-Ms group. Two deaths in the MTZ-I (600 mg/kg) group were identified. Changes in clinical biochemistry parameters were of minor nature and occurred in the absence of a clear dose-related distribution. Lymphocytes and leukocytes increased in the MTZ and MTZ-Ms groups, suggesting immunostimulation. Histopathological examination revealed hyperplasia of intestinal epithelium along with alterations in spleen and testis in rats treated with MTZ (600 mg/kg) and MTZ-Ms (1000 mg/kg). In MTZ-I groups, mortality was considered to be related to treatment with test substance in both acute and subacute toxicity assays. The results suggest that MTZ and MTZ-Ms have similar toxicological profile. However, further investigations are warranted.