β-Mannanases are involved in the conversion and modification of mannan-based saccharides. Using a retaining mechanism, they can, in addition to hydrolysis, also potentially perform transglycosylation reactions, synthesizing new glyco-conjugates. Transglycosylation has been reported for β-mannanases in GH5 and GH113. However, although they share the same fold and catalytic mechanism, there may be differences in the enzymes' ability to perform transglycosylation. Three GH5 β-mannanases from Aspergillus nidulans, AnMan5A, AnMan5B and AnMan5C, which belong to subfamily GH5_7 were studied. Comparative studies, including the GH5_7 TrMan5A from Trichoderma reesei, showed some differences between the enzymes. All the enzymes could perform transglycosylation but AnMan5B stood out in generating comparably higher amounts of transglycosylation products when incubated with manno-oligosaccharides. In addition, AnMan5B did not use alcohols as acceptor, which was also different compared to the other three β-mannanases. In order to map the preferred binding of manno-oligosaccharides, incubations were performed in H2 (18)O. AnMan5B in contrary to the other enzymes did not generate any (18)O-labelled products. This further supported the idea that AnMan5B potentially prefers to use saccharides as acceptor instead of water. A homology model of AnMan5B showed a non-conserved Trp located in subsite +2, not present in the other studied enzymes. Strong aglycone binding seems to be important for transglycosylation with saccharides. Depending on the application, it is important to select the right enzyme.