Raffinose family oligosaccharides (RFOs) are widespread across the plant kingdom, and their concentrations are related to the environment, genotype, and harvest time. RFOs are known to carry out many functions in plants and humans. In this paper, we provide a comprehensive review of RFOs, including their beneficial and anti-nutritional properties. RFOs are considered anti-nutritional factors since they cause flatulence in humans and animals. Flatulence is the single most important factor that deters consumption and utilization of legumes in human and animal diets. In plants, RFOs have been reported to impart tolerance to heat, drought, cold, salinity, and disease resistance besides regulating seed germination, vigor, and longevity. In humans, RFOs have beneficial effects in the large intestine and have shown prebiotic potential by promoting the growth of beneficial bacteria reducing pathogens and putrefactive bacteria present in the colon. In addition to their prebiotic potential, RFOs have many other biological functions in humans and animals, such as anti-allergic, anti-obesity, anti-diabetic, prevention of non-alcoholic fatty liver disease, and cryoprotection. The wide-ranging applications of RFOs make them useful in food, feed, cosmetics, health, pharmaceuticals, and plant stress tolerance; therefore, we review the composition and diversity of RFOs, describe the metabolism and genetics of RFOs, evaluate their role in plant and human health, with a primary focus in grain legumes.