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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. It has been confirmed the presence of microorganisms belonging to fungi, plant and bacteria kingdoms amplifying the rDNA in the characteristic regions (ITS, 18S, 16S) by performing PCR amplification with specific primers. For this purpose, two different biofilm sampling procedures, one completely non-invasive (swab in physiologic saline solution) and one micro-invasive (adhesive tape) have been employed: the adhesive tape performed better than swab in relation to DNA purity and quantity. After the biofilm characterization, the application of the phytochemicals occurred. For the realization of formulations, the EOs and their major components have been incorporated inside an innovative hydrogel matrix conceived for the cleaning of cultural heritage. The biocidal and cleaning action was evaluated with colorimetric measurements before and after the application of the products and, in the case of the surfaces treated with the essential oils, these measurements have been repeated 6 weeks after the end of the treatment. It emerged that the formulations containing the active phenolic compounds have, in most cases, a better efficacy against the multispecies biofilm. Anyway, it was verified that the biocidal action of the essential oils lasts in time, since colour variation towards the original colour of the surface have been determined 6 weeks after the end of the procedure. [ABSTRACT FROM AUTHOR]