Stone architectural heritage exposed outdoor represents a challenging habitat for biological growths; nevertheless, biocolonization on heritage structure is ubiquitous and represents a major mechanism of alteration. However, the identification of specific microorganisms with known reactivity towards the stone substrate does not necessarily imply that a biodeterioration process is in progress and, in specific conditions, bioprotection effects have been highlighted as a result of colonization. The main objective of the present research is to evaluate the biofilm formation on different lithotypes exposed to similar environmental polluted conditions, and to investigate whether the presence of subaerial biofilms can be associated to an increased magnitude of deterioration of the colonized surfaces with respect to the not colonized ones. In particular, the research examines the extensive biological colonization of the stone surfaces of the façade of the Cathedral of Monza (Italy). Four metamorphic stones widely used in the façade and showing rather different compositional, mineralogical and microstructural features were studied. The state of conservation of the stones was characterized under the mineralogical and compositional point of view by X-ray diffraction and Fourier Transformed infrared analysis. The microstructure of colonized substrates and of reference not colonized ones was studied by means of optical and electron microscopy, to comparatively evaluate the damage extent and weathering patterns in both conservative conditions. The structure and the architecture of biofilms growing on different lithic surfaces were investigated by CLSM in both fluorescence and reflection modes. Captured images were analyzed for 3D reconstructions of biofilm samples. The biovolumes were also calculated to estimate the total biomass. The results indicate that the four lithotypes showed different colonization extents. However, even in presence of extensive biological growth, chemical-physical deterioration mechanisms caused by environmental exposure were largely responsible for deterioration. A relationship between compositional and surface morphological features and biocolonization was also observed.