Probiotics are known for their health-promoting abilities through the production of various metabolites, and also benefits are membrane associated. Probiotic Enterococcus faecalis AG5 has been previously assessed for its probiotic properties and this study further strengthens that claim. Strain AG5 was inoculated with 0.3% sodium glychocholate (glycine conjugated) and bile salt mixture for different time periods. The percentage assimilation of cholesterol ranged from 14% to 33.1% in the bile salt mixture, but was slightly higher, ranging from 16.55% to 53.1%, in sodium thioglycocholate alone. Similarly, the bile salt hydrolysis activity was measured in terms of cholic acid release at different intervals. The overall range of cholic acid released ranged from 70.4 to 479 μg/mL. Fatty acid production is also an important criterion for a probiotic selection and it was found that the strain produced several long-chain fatty acids (LCFA) such as tetradecanoic acid, hexadecanoic acid, octadecanoic acid and bis(6-methylheptyl) phthalate. Besides LCFA, AG5 produced short-chain fatty acids (SCFA) such as propionic acid, which was estimated comparing liquid chromatography and mass spectrometry with standard SCFA. Thus the ability of deconjugation of bile salt and SCFA production is a value-added property of probiotic AG5, with the promise of being beneficial for human health. © FEMS 2019.