BACKGROUND Deterioration by microbial activity is common in the built cultural heritage, where phototrophic microorganisms are primary colonizers that form complex communities associated with heterotrophic microorganisms. Therefore, this article analyses the antiphototrophic effect of ZnO and CaZn2(OH)6·2H2O (CZ) nanoparticles (NPs) on three limestone lithotype coupons (Xcambo, red limestone and shellstone). The nanomaterials were obtained via sol–gel (SG), hydrothermal (HT) and mechanochemical (MS) routes and characterized using powder X‐ray diffraction and scanning electron microscopy. Two concentrations of NPs (5000 and 10 000 μg mL−1) were applied to the coupons; afterwards, phototrophic microorganisms isolated from Chichén‐Itzá, Yucatan were inoculated. The percentage of protection was calculated with ImageJ software, and chromatic parameters were measured to identify visible changes in the stones. RESULTS The results show differences in the protective effect and color variations depending on the nanomaterials and phototrophic microorganisms applied in the limestone coupons. CZ and ZnO protect more than 72% on the surface of shellstone and Xcambo except for ZnO‐SG in shellstone. The growth of Gloeocapsa sp. in red limestone with ZnO‐SG and CZ‐SG was equal to or greater than that of the control without NPs. In general, the NPs protect the limestone surface against phototrophic microorganisms up to 10% more than commercial algaecide. The coupons treated with ZnO‐HT and CZ‐HT have greater protection against biofilm formation than other coatings. CONCLUSIONS In general, the coatings show a higher antiphototrophic protection than the controls, mainly the CZ‐HT coatings, with better results at lower concentration. Therefore, these nanomaterials could be used to inhibit the formation of biofilms on built cultural heritage. © 2022 Society of Chemical Industry (SCI).