This study reports the effects of probiotics and alpha-tocopherol administration on microbial flora of rat gastrointestinal tract in a model system containing aspirin, ethanol and ammonia which are causative agent of gastric mucosal injury in animal and clinical studies. Sixty rats were used and randomly divided into three groups as Control (C), Probiotic mix culture (P) and alpha-Tocopherol (T). C, P and T groups received skim milk, probiotic mix culture and alpha-tocopherol for 14 days, respectively. Then, each group was also divided into four subgroups as aspirine, ethanole, ammonia and non-treated group. On day 15, aspirin, ammonia and ethanol were administrated to three subgroups, respectively. Non-treated subgroup just received saline in equal volume. In the 1, 5, 10 and 14th day of the feeding, fecal samples were taken from rats and lactic acid and coliform bacteria were determined. On day 15, rat intestine was taken out and examined for microbial flora. The results indicated that probiotic mix culture and alpha-tocopherol application had no significant effects on microbial flora of rat intestinal tract through 14 days of intake. In addition, there was not considerable difference in microbial flora in the jejunum, ileum and caecum for C, P and T groups with or without administration of aspirin, ethanol and ammonia.