Probiotics are live microorganisms which show beneficial effects for maintaining good health in human and animals. The role of probiotics as functional ingredients in food has been highly appreciated by the consumers in recent years. The objective of the present study was enzymatic profiling and biochemical characterization of the five probiotic yeast strains viz.Yarrowialipolytica VIT-MN01, Kluyveromyceslactis VIT-MN02, Lipomycesstarkeyi VIT-MN03, Saccharomycopsisfibuligera VIT-MN04 and Brettanomycescustersianus VIT-MN05. The probiotic yeasts were tested for fermentation ability of carbohydrates viz. glucose, fructose, lactose, sucrose, dextrose, galactose, starch, mannitol, arabinose, xylose, cellulose, cellobiose and assimilation capacity of nitrogen. Among the five probiotic yeast strains, S. fibuligera VIT-MN04 was the most capable strain to ferment all the carbon sources except galactose and could use all the nitrogen sources. Further, yeast strains were tested for the production of the enzymes such as oxidase, catalase, protease, amylase, esterase and L-asparaginase. They were also tested for the ability of glycerol metabolism.Among the five yeast strains,S. fibuligera VIT-MN04 showed significant enzymatic and biochemical activities. Therefore, S. fibuligera VIT-MN04can be chosen as potential probiotic which can be used for further studies.