Biodeterioration poses a major challenge to the conservation of stone monuments, particularly in UNESCO World Heritage sites like the Convent of Christ and the Batalha Monastery in Portugal. This study introduces novel sampling locations in both monuments, allowing a more targeted characterization of the eukaryotic microbial communities colonizing the Convent of Christ's stone surfaces. Microbiota distribution across distinct zones revealed that key lichenized fungi with biodeteriogenic potential—Dirina, Xanthoria, Purpureocillium, Verrucaria, and Cystocoleus—were predominant, as identified by Next Generation Sequencing (NGS). Additionally, an orange-pink bacterial biofilm from the Batalha Monastery was analyzed through colorimetric techniques and antimicrobial assays, demonstrating that Biotin-T was the most effective treatment, inhibiting bacterial growth while preserving the stone's original appearance and progressively reducing microbial viability over time. These findings provide insights into biodeterioration processes and offer potential solutions for the conservation of historic stone heritage. Keywords: Biodeterioration; Environmental DNA; NGS; Stone monuments; Pink pigmentation; Antimicrobial assays; Heritage preservation.