Ancient books and documents constitute an important cultural heritage, which are composed by different supports, such as cardboard, parchment and paper. Due to their composition (animal- and plant-based matrices), they allow bacteria and fungi to thrive, causing the phenomenon of biodeterioration, an ecological succession in parchment. Four ancient books called “Compositionum” from the Apostolic Vatican Archive, made of the same materials, exposed to weather-beating conditions and showing different degrees of deterioration, were analysed by a multidisciplinary approach: DNA metabarcoding using NGS, Light Transmission Analysis and Raman and FTIR spectroscopy. The results highlighted how the biodeteriogen community composition changed from the least to the most damaged, without evidence of significant microbial transfer across the three matrices. The results allow confirmation of the ecological succession as biodeterioration process, including cardboard and paper, in addition to in parchment. These results give important insight for the conservation and restoration practices of all matrices.