The Stable Isotope Unit of the National Centre of Scientific Research and the Ephorate of Kavala and Thasos collaborated on a project to investigate construction materials from the Ancient Theatre on Thasos Island, which was funded by NSRF European funds (2007-2013 and 2014-2020). Bulk samples of marble were collected to determine their provenance, technology, degradation features, and mechanical and physicochemical properties, with the ultimate goal of developing a conservation plan. By utilizing novel scientific techniques, the research team was able to date constructions discovered within excavations and establish remedial and preventive conservation plans using marble technology. The samples were characterized in terms of their isotopic, chemical, and mineralogical composition. Additionally, extended physicochemical and mechanical testing was applied to estimate the current condition of the Ancient Thasos Theatre. The 13C and 18O isotopic composition of the calcitic marbles was analysed to determine the environmental conditions during calcite formation, making it possible to define the marble provenance. Compositional and morphological analyses were achieved using energy-dispersive X-ray analysis by scanning electron microscopy, while the mineralogical phases were detected using X-ray diffraction and petrographic (polarized optical microscopy) analysis. Mechanical testing according to European Standards was achieved through the examination of compressive tensile strength and grain size distribution.