Glycosylhydrolases of various origins were used to produce fucose-containing disaccharides with prebiotic potential using different donor substrates and l-fucose as the acceptor substrate. Eight different disaccharides were synthesized as follows: three -d-galactosyl-l-fucosides with glycosidase CloneZyme Gly-001-02 using d-lactose as a donor substrate, two with a structure similar to prebiotics; one -d-galactosyl-l-fucose with -d-galactosidase from Aspergillus oryzae using d-lactose as a substrate donor; and four -d-glucosyl-l-fucosides with -d-glucosidase from Saccharomyces cerevisiae using d-maltose as a donor substrate. All disaccharides were purified and hydrolyzed. In all cases, an l-fucose moiety was present, and it was confirmed for -d-galactosyl-l-fucose by mass spectrometry. High concentrations of l-fucose as the acceptor substrate enhanced the synthesis of the oligosaccharides in all cases. The three enzymes were able to synthesize fucose-containing disaccharides when l-fucose was used as the acceptor substrate, and the highest yield was 20% using -d-galactosidase from Aspergillus oryzae.