Previous studies have shown the interest in using Atomic Force Microscopy (AFM) to investigate the parietal mechanical properties of hemp or flax fibres, in correlation with their structure or maturity (1–3), especially in PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) mode. However, this technique is relatively new, and its application is often limited to the engineering and biological fields, but the small quantity of the sample required make it a useful tool also to study materials from the cultural heritage. In this research, two yarns of few centimetres were taken from the back of four Italian paintings of the art gallery of Ascoli Piceno, dated around 1600-1700, and one yarn of each pair was selected for the AFM mechanical characterization. PF-QNM analysis was performed with a Multimode AFM (Bruker Corporation, USA) equipped with a RTESPA-525 probe (Bruker Corporation, USA) and a maximum load of 200 nN; the cantilever was carefully calibrated using the Sader method and the indentation modulus of Highly Oriented Pyrolytic Graphite (HOPG) was taken as reference.