Calixarene–iminosugar derivatives bearing four 1-deoxynojirimycin units at the upper or lower rim of calix[4]arenes in a fixed cone conformation were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and their inhibitory activity was evaluated against five glycosidases. Modest but significant affinity enhancements of up to seven per 1-deoxynojirimycin unit over the monovalent iminosugar derivative were observed for the inhibition of Jack bean (Canavalia ensiformis) α-mannosidase. It was also demonstrated that the residual copper ions did not contribute to the inhibitory properties of the newly prepared calixarene-based multivalent iminosugars.