Prebiotic is classically defined as a nondigestible food element that selectively promotes the growth and functionality of the colon microflora and eventually improves host health. It mainly consists of carbohydrates like oligosaccharides, various peptides, and also dietary fibers. Prebiotics have been widely used as nutraceuticals having essential roles in the enhancement of gut microbiota and also work as therapeutics against various inflammatory and infectious diseases associated with the gut. The objective of this chapter is to focus on the biochemical characteristics of various types of prebiotics that are very much essential in our daily diet and their use as nutraceuticals to promote the growth of beneficial microbiota to protect the gut epithelial and mucosal layer against the damage from invading pathogens and toxins. It has been found that the use of various prebiotics as a supplementary diet in both human and animal models significantly regulates the growth of Bifidobacterium and Lactobacillus and increases the fermentation within the gut leading to the generation of SCFAs and immunomodulation of the host immune system. It is used as a therapeutic agent for the inflammatory bowel disease (IBD) and also inhibits the progression of cancer. Furthermore, it also increases the efficiency of digestion and absorption of nutrients and also protects against pathogenic infections.