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In 2006, after a visual inspection of the Leonardo da Vinci's Atlantic Codex by a scholar, active molds were reported to have been present on Codex pages showing areas of staining. In the present paper, molecular methods were used to assess the current microbiological risk to stained pages of the manuscript. Bacterial and fungal communities were sampled by a non-invasive technique employing nitrocellulose membranes. Denaturing gradient gel electrophoresis of 16 S rRNA gene and internal transcribed space regions were carried out to study the structure of the bacterial and fungal communities and band patterns were analyzed by the multivariate technique of principal component analysis. Any relationship between the presence of an active microbial community and staining was excluded. The presence of potential biodeteriogens was evaluated by constructing bacterial and fungal clone libraries, analyzing them by an operational taxonomic unit (OTU) approach. Among the bacteria, some OTUs were associated with species found on floors in clean room while others were identified with human skin contamination. Some fungal OTU representatives were potential biodeteriogens that, under proper thermo-hygrometric conditions, could grow. The retrieval of these potential biodeteriogens and microorganisms related to human skin suggests the need for a continuous and rigorous monitoring of the environmental conditions, and the need to improve handling procedures.