Endoglucanase (EG) is a key enzyme during enzymatic preparation of cellulose nanocrystals (CNCs).Myceliophthora thermophilais a thermophilic fungus that has thermal properties and a high secretion of endoglucanases (EGs), and could serve as potential sources of EGs for the preparation of CNCs. In this work, four different GH families (GH5, GH7, GH12, and GH45) of EGs fromM. thermophilawere expressed and purified, and their enzymatic characteristics and feasibility of application in CNC preparation were investigated. It was shown that the MtEG5A fromM. thermophilahas good potential in the enzymatic preparation of CNCs using eucalyptus dissolving pulp as feedstock. It was also observed that there was a synergistic effect between the MtEG5A and other MtEGs in the preparation of CNCs, which improved the yield and properties of CNCs obtained by enzymatic hydrolysis. This study provides a reference for understanding the enzymatic characteristics of different families of EGs fromM. thermophileand their potential application in nanocellulose production.