Galactooligosaccharides, a group of oligosaccharides that are classified as prebiotics due to their structure and indigestibility in the upper part of the digestive system, are increasingly being studied. Their prebiotic effect is most pronounced in the lower part of the digestive system—in the colon—since they become food for beneficial bacteria there, thereby promoting their growth. Beneficial bacteria such as bifidobacteria and lactobacilli are needed in large numbers due to their positive effect on the intestinal microflora. By chemical structure, these are compounds consisting of a chain of galactose molecules linked to a glucose molecule by glycosidic bonds. The aim of this work is to explore the synthesis of these compounds from various substrates, specifically lactose, and to investigate their potential applications. The synthesis of galactooligosaccharides is carried out using enzymes like β-galactosidase, which break down lactose (a disaccharide consisting of glucose and galactose) and then catalyze the transfer of galactose units to different acceptor molecules. As prebiotic compounds, they are primarily used in the food industry, and also in the pharmaceutical and cosmetic industries. Additionally, they help in maintaining bone health and strengthening the immune system.