Fructooligosaccharides (FOS) are bioactive compounds of great interest in the food and pharmaceutic industry for their multiple functional properties that include prebiotic activity, immunomodulation, and improvement of mineral absorption, preventing the development of chronic diseases such as colon cancer and irritable bowel disease. FOS are present in low amounts in vegetal sources varying in concentration and type depending on the source and time of the year. FOS can be produced by microorganisms with the enzymatic capacity to synthesize FOS parting from sucrose or by hydrolysis of inulin or levan. Microbial production of FOS has undergone many advances in the recent decade to improve FOS yield of production, productivity, and FOS purity. Production strategies include enzymatic synthesis, whole-cell fermentation, successive fermentations, co-cultures, genetically modified strains, and new strategies employing alternative substrates taking advantage of agro-industrial residues or sucrose-rich feedstocks. The most relevant strategies for the microbial production of FOS will be reviewed in this chapter.