This laboratory research aimed at characterizing the microorganisms that colonize the limestone walls of the Unfinished Sacristy of the Convent of Christ (Tomar, Portugal), as well as to study their control by means of biocidal agents. Six different locations were sampled through swabbing and the microorganisms were characterized using classical microbiology techniques. The presence of fungi, heterotrophic bacteria and autotrophic microorganisms was verified in all samples. The relative effectiveness of different biocides was tested through the analysis of the inhibition halos they caused in the growth plates of bacterial and fungal isolates. For bacteria, two currently used biocides were tested: Biotin T and benzalkonium chloride. For fungi, these same two biocides plus a paint additive were assessed. The biocides showed different levels of effectiveness for different microorganisms: the benzalkonium chloride was more effective on bacterial isolates, while Biotin T showed a better performance on fungal isolates.