The degradation of stone materials depends on several interlinked factors. The effects caused by biodeteriogens on mineral-based substrates are now increasingly considered in the field of cultural heritage conservation from different experimental approaches. In this study, biodeteriogenic micro- and macroflora within the gothic building of Santa Maria della Pietà, Squillace, Calabria, have been analyzed using multiple approaches, such as optical microscopy and molecular techniques. All 17 plant species detected are usually widespread in Mediterranean regions and some of these, such as Ailanthus altissima and Ficus carica, showed a very high hazard index, which is potentially dangerous for masonry stability. Fungi, cyanobacteria, and green algae were identified within biofilm compositions in a total of 23 different taxa, showing many similarities with microbial associations commonly found in cave and hypogean environments. All of the 11 fungal taxa detected belong to Ascomycota phylum, with Penicillium as the most represented genus. Photoautotrophic organisms are mostly represented by filamentous genera, with widespread presence of Leptolyngbya as the most abundant genus. The results highlighted how the singular environmental conditions of the study site, combined with the architectural features and the building materials, determined all the degradation phenomena affecting the building’s internal surfaces, compromising over time the structural integrity.