Nutrients-enriched diet is a vital need for the survival of living organisms, as it is shown by several research studies that the nutrient-deficient diet may cause many diseases, which can be manipulated with the adequate intake of different dietary substances. Several modifications have been made in the dietary pattern with great interest towards the intake of prebiotics particularly fructooligosaccharides (FOS) which plays a significant role in the growth of gut microbiota in different ways. Oligosaccharides have low-calorific values with non-carcinogenic properties that helps in the absorption of ions with the reduction in the cholesterol level. These functional components are Generally Regarded as Safe (GRAS) for consumption, which can be extracted from different agricultural byproducts (coffee byproducts, sugarcane byproducts, cereal brand, etc.) These agri-industrial substrates are considered as one of the economical approaches used in the production of low-cost fructooligosaccharides. The innovative and novel prebiotics have been gaining the attention of health-conscious consumers exploited from agri-food waste biomass. This chapter deals with the structures of different FOS, their properties, different methodologies for the production of microbial FOS with or without enzymes and recombinant technologies using agri-food wastes or by-products, and their industrial applications in the food sector.