The metabolic capacity of the intestinal microflora and its influence on the human body are not fully understood. One area of interest is the metabolism of drugs used to treat disease. To gain a better understanding, major genera present in the intestine were tested for their ability to metabolize phenobarbital, a pharmaceutical drug used to treat epilepsy. We tested Bifidobacterium, Bacteroides, Enterococcus, Eubacterium, Clostridium, and Staphylococcus. Growth analysis at different concentrations of phenobarbital and gas chromatography-mass spectrometry analysis were used to determine the response and metabolism of phenobarbital. Our growth curve results demonstrated that the lag time for some bacteria was affected in the presence of high concentrations of phenobarbital. The generation time was also affected based on the concentration and specific bacterial type. Finally, it was shown that Bifidobacteirum adolescentis and Bifidobacterium bifidum were unique in metabolizing phenobarbital (reductively cleaved) into the compound alpha-ethyl-benezeneacetamide, which was an unexpected metabolite. The data demonstrate for the first time the ability of Bifidobacterium to metabolize phenobarbital into the potentially toxic metabolite alpha-ethyl- benezeneacetamide. The consequences of the generated metabolite may affect the surrounding intestinal bacterial population or the effectiveness of the drug in treating epilepsy.