Microbial enzymes find application in enzymatic reactions for the production of fructooligosaccharides (FOS). Demands for an alternative healthy sweetener and multifunctional FOS have prompted investigators to explore microorganisms for microbial enzyme production as well as to develop bioprocesses for the production of high-fructose syrup and oligosaccharides based on transfructosylation of sucrose or hydrolysis of inulin. Fructosyltransferases have been characterized in several molds, yeasts, and a few bacteria. Recently, there has been a growing interest in finding novel enzyme producers by using agroindustrial media in both submerged and solid-state fermentation. This review discusses current knowledge on production, properties, and applications of microbial enzymes involved in the production of fructooligosaccharides and considers recent advances in the field. It also describes a comprehensive and illustrative analysis on basic and applied aspects of fungal enzymes, different substrates of microbial growth, and fundamental biotechnological and catalytic aspects to illustrate the potential of these biocatalysts in the food and bioprocessing industries.