A novel high level oriented assembly of zinc hydroxide carbonate nanocrystals in chitosan was fabricated via a hydrothermal process. Hydrated chitosan acts as a template supporting and structure-directing reagents for the formation and assembly of zinc hydroxide carbonate nanocrystals. Zinc hydroxide carbonate nanocrystals were heterogeneously nucleated at the interface of hydrated chitosan crystallization regions through hydrogen bonding, lattice geometrical matching, and stereochemical matching interactions with ordered chitosan chains. The products obtained at different reaction stages were characterized with SEM, TEM, XRD, XPS, and TGA. The antifungal activity assays of CZHC, zinc hydroxide carbonate, chitosan, chitosan simply blended with zinc hydroxide carbonate (1:1), and nano zinc oxide were investigated. The study exhibited that the CZHC nanocrystals showed an antifungal activity against Cotton Verticillium, Rhizopus, and Mucorales and showed a better antifungal activity than other test samples against Rhizopus.