Ancient parchments are commonly attacked by microbes, producing purple spots and detachment of the superficial layer. Neither standard cultivation nor molecular methods (DGGE) solved the issue: causative agents and colonization model are still unknown. To identify the putative causal agents, we describe the 16 S rRNA gene analysis (454-pyrosequencing) of the microbial communities colonizing a damaged parchment roll dated 1244 A.D. (A.A. Arm. I-XVIII 3328, Vatican Secret Archives). The taxa in damaged or undamaged areas of the same document were different. In the purple spots, marine halotolerant Gammaproteobacteria, mainly Vibrio, were found; these microorganisms are rare or absent in the undamaged areas. Ubiquitous and environmental microorganisms were observed in samples from both damaged and undamaged areas. Pseudonocardiales were the most common, representing the main colonizers of undamaged areas. We hypothesize a successional model of biodeterioration, based on metagenomic data and spectroscopic analysis of pigments, which help to relate the damage to a microbial agent. Furthermore, a new method (Light Transmitted Analysis) was utilized to evaluate the kind and entity of the damage to native collagen. These data give a significant advance to the knowledge in the field and open new perspectives to remediation activity on a huge amount of ancient document.