Travertine is a sedimentary carbonate rock widely used in Roman arts and frequently subjected to biodeterioration. In this preliminary multi-analytical study, three essential oils (EOs) from T. vulgaris, O. vulgare, C. nepeta and their respective phenolic major components (thymol, carvacrol, pulegone) were selected to evaluate their biocidal potential against a multi-species biofilm grown on a travertine test wall located inside Sapienza University Campus in Rome. A preliminary characterization of the biofilm occurred through the employment of genomic methodologies.