The importance of stone colonisation by microorganisms has led to an extensive literature on mechanisms and rates of physicochemical degradation of stone surface, both in laboratory and field contexts. Biological colonisation of a stone surface depends on intrinsic stone parameters like mineral composition, texture, porosity, and permeability, as well as on environmental parameters. In the present study, quantification of stone surface roughness and its relationship to epilithic colonisation was demonstrated for three types of limestones throughout non-destructive techniques, namely optical surface roughness instrument and digital image analysis. According to the roughness average (Ra) and mean roughness depth (Rz) determined for Ançã limestone, Lioz limestone and Lecce stone, it can be concluded that great surface roughness stones render them prone to microbial colonisation.