In historic mortars, the main binder used are calcitic or dolomitic lime. However, these mortars are also susceptible to the biochemical action of fungi causing undesirable changes in those materials. Nanotechnology has been investigated as a strategy to mitigate the deterioration of the cultural heritage. Photocatalysis has proven effective as an agent of degradation of organic contamination by inhibiting the recolonization of substrates affected by biological growth. This study verifies the effectiveness of a nanometric photocatalyst (nTiO2-P25, 3% in deionized water) sprayed on the surface of simple lime–sand mortar (1:4 mass trace), typically used in historical buildings, against the growth of Aspergillus niger fungal isolates, inoculated with a 106 spore/mL suspension. The samples were exposed to the incidence of artificial radiation of UVA light with a wavelength of 285 nm to activate the nTiO2. We observed a reduction in fungal colonies, especially after four hours of exposure per day, reaching 100% in the staining reduction in one case. Another aspect observed was that the previous application of nTiO2 on the mortar samples caused alterations in the morphology of the A. niger fungus structures, evidence of a disturbance in the growth of the colonies or even their reduction over time