Prebiotics, including both natural and synthetic non-digestible carbohydrate substances, have gained significant attention for their ability to support the proliferation of beneficial gut microbes. Among these, fructooligosaccharides (FOS) play a crucial role as substrates within the digestive system, aiding in producing of shortchain fatty acids by beneficial microorganisms, thus promoting their growth. The benefits of prebiotics in modulating gut microbiota, enhancing mineral absorption, and managing conditions such as diabetes, allergies, and metabolic disorders have led to increased interest in their applications in the pharmaceutical and food industries, with some products already available as supplements. Biosynthesis of FOS not only takes place in plant systems but enzymes responsible for the synthesis of these oligosaccharides are present in fungi and bacterial cells. Characterization of FOS mainly molecular weight and structural identification can be done with spectroscopic and chromatographic techniques. Modulation of the gut microbiome through prebiotic interventions is also reported. This comprehensive review focuses on fructooligosaccharides, highlighting their evolving roles in dietary interventions and potential future research directions. By addressing key challenges and exploring possible advancements, this review seeks to contribute to the ongoing efforts to understand and harness the therapeutic potential of prebiotics for human health.