Abstract Nucleotide-activated di- and oligosaccharides represent a novel class of glycoconjugates. They are components of human milk with still unknown biological function. Synthetic access to a wide range of nucleotide di- and oligosaccharides would also facilitate their utilization as donor substrates or inhibitors of Leloir-glycosyltransferases. We here present for the first time the synthesis of β1-3-linked nucleotide activated disaccharides by recombinant β3-galactosidase C from Bacillus circulans. UDP-Glc, UDP-GlcNAc, and UDP-GalNAc reacted as acceptor substrates in the transglycosylation reaction with p-nitrophenyl-β-galactoside as donor substrate. In an attempt to optimise the transglycosylation reaction, focused microwave irradiation was investigated. In comparison to conventional thermal heating product compositions and product yields were affected by microwave irradiation and depended on the used acceptor substrate. Microwave irradiation was advantageous for syntheses with UDP-GlcNAc as preferred acceptor substrate of β3-galactosidase C. The β1,3 linked UDP-disaccharide was the main product with minor fractions of UDP-tri- and UDP-tetrasaccharide. In summary, access to important UDP-disaccharides such as UDP-LacNAc type 1 and UDP-Thomsen-Friedenreich(T)-antigen was accomplished for further studies of their role as donor substrates or inhibitors of glycosyltransferases.