The Eugénio de Almeida Foundation’s Casa de Fresco is a historical monument of valuable historic–artistic significance, which currently reveals an assortment of biofilms due to the proliferation of microorganisms in the stone and rocaille elements. The biodeterioration in this area was studied as part of the Conservation and Restoration Project. We effectively characterized the local microbial community using modern high-throughput DNA analysis. Our results suggested the existence of a variety of lichens or lichenized fungi, including genera such as Variospora, Verrucaria, Circinaria, and Caloplaca. Furthermore, we detected several prokaryote microorganisms related to the identification of these lichens. To properly deal with this microbiological issue and avoid fungal recolonization, we evaluated available commercial antimicrobial treatments. Keywords: stone monuments; patrimony biodeterioration; metagenomic approach; lichenized fungi; antimicrobial activity; heritage conservation.