The thallus–substrate relationship of Tephromela atra var. calcarea was investigated to determine whether the colonisation of carbonate-rich rock can be related to a “superficial decalcification” of the substrate, as claimed by some authors. Fragments of thalli still adhering to the substrate from the TSB herbarium were embedded in epoxy resin to obtain cross-sections, which were analysed by FPA-FTIR microspectroscopy in reflection mode to acquire chemical imaging data reflecting the spatial distribution of molecular components. The cross-sections were then stained with periodic acid-Schiff, and the percentage of hyphal spread was measured in selected areas of 2 mm2 at fixed distances along vertical transects from the thallus–substrate interface to the hyphal-free substrate. X-ray diffraction (XRD) was performed on additional fragments to detect any biomineralization products present. The hyphae of T. atra penetrated all calcareous substrates to a maximum depth of 0.8 mm, also piercing single calcite clasts. Hyphal spread varied greatly between substrates, with a minimum in compact limestone and a maximum in porous limestone. XRD analyses showed the presence of the biominerals whewellite and weddellite in varying amounts, and confirmed the presence of calcite in all samples, except in one occurring on Roman brick. High-resolution FTIR chemical maps showed the presence of calcite in medium/high to high concentration at the thallus–substrate interface. No evidence of calcite depletion was observed. These results do not support a significant carbonate depletion of the surface of the carbonate-rich rock colonised by T. atra, whose hyphae can actively penetrate the calcite clasts. Keywords: Biodeterioration; Calcite; Carbonate-rich substrate; Oxalates; FTIR; XRD.