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There is much interest in the study and production of nondigestible oligosaccharides (NDOs), due to their bioactivities and beneficial effects to the human health. The main approach in the production of NDOs relies on the action of glycosidases performing hydrolysis or transglycosylation of polysaccharides and sugars. In this chapter, a description of the main microbial glycosidases used for NDOs production, their sources, their principal properties, and a description of the production processes with the better results obtained are discussed.