Natural stones have been used throughout ages to raise buildings, monuments and sculptures. The choice of stones was often more dictated by their aesthetics and availability rather than by their intrinsic properties. Moreover the stone is one of the most durable materials but despite of its ever lasting renown as it goes through many centuries, it is affected by the weathering. Nowadays, the historical and cultural significance of many monuments calls to its preservation. Stone deterioration was enhanced due to the environmental pollution that did not exist in former times. It is the combination of environmental factors comprising climatic conditions (temperature, humidity) and anthropic activities (air pollution) and leads to physical, chemical weathering and biological development. The colonisation of stone by micro-organisms causes on first an aesthetic effect by a fouling and a change of stone colour. Over time the living of organisms induces undesired change of properties by a biochemical action made by the cell metabolism and a mechanical action by the growth of the biomass. Consequently, that leads to a weakness of the stone and its breakdown. Biofilm is defined as a microbial community and is composed of water, microbial cells and extracellular polymeric substances. It can be observed on any solid surface in which sufficient moisture is available in nature. These community provides a good medium to the development of macroscopic vegetation which makes irreversible and physical damage in stone by roots. Detecting as soon as possible biofilms and before the greening therefore before the fouling of stones is important in the conservation of Cultural Heritage. Indeed, that allows to limit the deterioration of monuments and to decrease the cost of the stone restoration. Non-destructive technique as colorimetry quantifies the colonisation of stone by the change of colour as the greening but the settlement of pioneer bacteria can’t be detected by this method because they are not colourful. Infrared thermography (IRT) has been used as a non-destructive technique to detect pioneer biofilms on stone. Experiment was displayed on a limestone called “Courville stone” which is used in major buildings in the surroundings of Paris. Stone samples have been exposed in an outdoor test to favour the natural colonisation by bacteria. Then three samples have been collected after few months and compared to three non-colonised sterile stones thank to stimulated Infrared Thermography. This technique assesses the thermal response of the sample when a flux of photons, emitted by a short ring flash, excites it. The absorption of photons increases the temperature in the vicinity of the lighted area comprising the sample surface that is recorded by a bolometer camera (FLIR SC655). Subsequent mathematical treatments of the recording has been carried out with a specific software (IR Exploreur) and in the aim at highlighting thermal variations. As a result, the settlement of the biofilm on stone is detected thank to several treatments as SVD decomposition.