Bile acids are synthesized from cholesterol and play an important role in regulating intestinal microflora. The different degrees of hydrophobicity and acidity of individual bile acids may affect their antimicrobial properties. We examined the antimicrobial effects of different bile acids on various microorganisms in vitro and confirmed whether these remain consistent in vivo. Using human bile acids, including ursodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, a disc diffusion test was performed, and a rodent model was created to determine the antimicrobial effects of each bile acid. The fecal bacterial population was analyzed using a real-time polymerase chain reaction. Each bile acid showed different microbial inhibitory properties. The inhibitory activity of bile acids against microbiota which normally resides in the gastrointestinal tract and biliary system, was low; however, normal flora of other organs was significantly inhibited. Changes in microbial counts after bile acid administration in a rodent model differed in the colon and cecum. The in vivo and in vitro results show that the antimicrobial effects of bile acids against intestinal microbiota were similar. In conclusion, bile acids could be a novel treatment strategy to regulate gut microbiota.