As a result of the research described in this article a safe, bio-based procedure has been established to treat hard-to-remove coherent deposits composed of gypsum, weddellite, calcium carbonate, apatite, nitrate, and aged proteinaceous matter from the wall paintings of the lower loggia of the Casina Farnese (Palatine Hill, Rome). Following a laboratory screening, three bacterial non-spore-forming strains were selected from our laboratory collection to solubilise calcium sulphate and carbonate (Cellulosimicrobium cellulans TBF11 E ), to degrade proteins (Stenotrophomonas maltophilia UI3 E ), to solubilise inorganic compounds and to degrade protein material (Pseudomonas koreensis UT30 E ). The living bacterial cells were suspended in a Laponite gel, which is highly compatible with the survival of the cells and is also easily applied, and removed from, vertical walls. Compresses containing microorganisms (micro-packs) were used in a series of biocleaning tests carried out in situ, from July 2012 to February 2013, in temperatures ranging from 6 °C to 37 °C. Each micro-pack contained a single bacterial strain (TBF11 E , UI3 E or UT30 E ). The micro-packs containing the different bacteria were applied, individually or in succession, depending on the nature and layers of the deposits to be removed. Contact times of between 24 and 48 h were established in accordance with the advice of the restorers. The strain TBF11 E removed the inorganic darker layer, UI3 E dissolved the brownish layer (probably aged casein) and UT30 E removed the mixed deposits. No residues were left after the treatment and the restorers successfully completed the restoration.