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This work presents the use of non-destructive techniques as testing tools for the diagnostic study of structural materials. Their contribution is essential for the successful design of necessary conservation interventions on monuments, which in this case are the Acropoles of Sarantapicho and Erimokastro at Rhodes, Greece. The diagnostic study of the masonry's structural materials was carried out using the Non-Destructive Technique of Ground Penetrating Radar, supported by an array of instrumental characterization techniques which included polarized optical microscopy, X-ray diffraction, scanning electron microscopy, mercury porosimetry, conductivity measurements and mechanical testing to characterize the structural materials and their decay products. With the exception of the off-white fossiliferous limestone (6 %) at Erimokastro, the other building materials examined presented a low porosity, below 1 %. In all type of building stones extensive biodecay products of grey-white or orange color that is attributed to lichen formation and cracks and detachment of stone material were observed. The biodecay is attributed to the extensive fungi growth that results in coloring of the stone (dark greyblack), pitting and microcracking, and to the conglomerates of dust particles consisting of pollen grains. The dark brown inclusions present intense surfacial cracks, pitting due to biodecay and extensive fungi micelle growth that penetrate vertically towards the main body of the stone. Moreover, the off-white fossiliferous limestone presents alveolar decay due to salt crystallization at the interior of the stone. Concluding, the use of non-destructive techniques in conjunction with instrumental characterization methods, allowed the successful assessment of the decay status and the mapping of the decay phenomena on the structural stones of the walls of the Acropolis at Ialyssos, and the Acropolis at Erimokastro.