In this paper, a new bio-cleaning procedure based on the glucose oxidase (GOx) has been applied on the travertine and peperino substrata to remove the biological patina (i.e., biofilm). Glucose oxidase, used as a model enzyme system, is able to produce in situ H2O2 (the cleaning agent having oxidizing properties) by the enzymatic reaction at room temperature. The travertine and peperino samples came from the Villa Torlonia in Rome (Italy), and an analytical diagnosis on them was performed applying several analytical techniques, such as the differential interference contrast microscopy (DIC), the optical microscope (OM), the Fourier transform infrared spectroscopy (FT-IR) and the X-ray fluorescence (XRF) that evidence the presence of biofilms on the substrata. Better results were obtained on the travertine samples in terms of the cleaning efficiency and the absence of the etching effect on the surface, eventually induced by the peroxide molecule. These results could be explained in terms of the different porosities of the two kinds of stone materials, according to the BET data. A comparative study was also performed to validate the new bio-cleaning procedure, using both traditional approaches based on saturated (NH4)(2)CO3 solution and EDTA in buffer solution and the enzyme lipase treatments. Among all, the cleaning procedure via GOx shows the best result, probably because the enzyme controls the concentration of the H2O2 in situ and also retains the H2O2 preferentially on the surface (where the biological patina is present) depending on the porosity of the substrata. A synergistic effect, with other enzymes such as lipase and protease, combined with the biocompatibility of the enzymatic treatments, could represent a new way for a higher cleaning efficiency to apply on different stone substrata. (C) 2010 Elsevier B.V. All rights reserved.