Oligosaccharides are carbohydrates, composed of up to twenty monosaccharides linked by glycosydic bonds, widely used in food and pharmaceutical industries. These compounds can be obtained by extraction from natural sources (milk, vegetables, fruits), and by chemical or biotechnological processes. In the last case, chemical structures and composition of the generated oligosaccharides depend on the type and source of enzymes, and on process conditions, including the initial concentration of substrate. Among the various functions of nondigestible oligosaccharides, one that has attracted attention is its prebiotic potential. The intestinal benefits of prebiotics, such as fructooligosaccharides and inulin as well as their symbiotic association with probiotic bacteria, encompass prevention and treatment of infectious diseases, including viral or bacterial diarrhea, and chronic inflammatory diseases such as ulcerative colitis. Other benefits attributed to prebiotics and probiotics include treatment of inflammatory intestinal and irritable bowel syndrome, prevention of cancer, and modulation of the immune system, mineral absorption and lipid metabolism. Fructooligosaccharides (FOS), galactooligosaccharides (GOS) and chitooligosaccharides (COS) have been widely studied for their prebiotic properties. Moreover, novel oligosaccharides with potential prebiotic activity are currently under investigation. This review will focus mainly on the biotechnological production, health benefits and applications of nonnatural oligosaccharides in the food industry.