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The evaluation of the antifungal activity of Mg(OH)2 and Ca(OH)2 nanoparticles (NPs), synthesized by sol–gel method and their mixtures at different concentrations, is reported. The antifungal activity of the hydroxide NPs was studied using Aspergillus niger and Penicillium oxalicum isolated from stone surfaces. These model organisms were selected due to their ability to grow on outdoor and indoor climates and their significant impact on human health. Moreover, the antifungal activity of Mg(OH)2 and Ca(OH)2 NPs dispersed in positively charged polymeric matrices based on partially quaternized poly(2-(dimethylamino ethyl) methacrylate) (pDMAEMA) was studied. With respect to the morphology, particle size, and textural properties of the NPs, the mixtures of Mg–Ca hydroxides revealed a uniform and smaller particle size, along with a greater surface area, as compared to pristine Ca(OH)2 NPs. However, the Ca(OH)2 and a mixture of Mg(OH)2 and Ca(OH)2 (10:90 weight ratio) NPs, showed an enhanced growth inhibition of A. niger and P. oxalicum, suggesting that the effect of particle size on the antifungal activity would not be a preponderating factor. In addition, improved antifungal properties against A. niger and P. oxalicum were detected in composite coatings based on hydroxide NPs dispersed in quaternized p(DMAEMA-co-METAI). The use of these systems might provide promising composite materials with potential antifungal properties for various applications.