The suitability of image analysis by colour-based pixel classification to quantify lichen colonization on the surface of and within marble, travertine and mortar stonework has been investigated. High resolution images of lichenized stonework surfaces were acquired at different field sites using a scanner, thus avoiding invasive surveys, and the percentage cover of lichen species was subsequently measured in the laboratory using dedicated software. Furthermore, microphotographs of polished cross-sections of lichenized marble, travertine and mortar, stained using the periodic acid-Schiff (PAS) method to visualize hyphae, were produced by the same software to quantify hyphal spread within the substratum, a parameter which can be used more successfully than the commonly used depth of hyphal penetration to quantify how much the lichen has affected the conservation of a stone substratum. Significant statistical differences in hue, saturation and intensity (HSI) of the lichen thalli and PAS-stained hyphae, with respect to the lithic substrata, allowed the software to discriminate and quantify the lichen species cover on, and hyphal spread within, the three investigated lithotypes. Since such a quantitative approach highlights the volume of influence of lichens on stonework, where bioweathering processes are likely to develop, it could be used to support decisions on the preservation of our stone cultural heritage.