Abstract This paper represents an interdisciplinary research focused on the microscopic and mineralogical investigations of the original fragments of a precious medieval painting, a rare byzantine Dormition of the Virgin in Italy. The painting has discovered in the rural monastery of Santa Maria del Rogato (Sicily), thanks to a recent restoration work. In order to obtain an exhaustive description of both the original mortars and pigments, several investigation methods are employed, from optical, polarization-fluorescence and electron microscopies, elemental analyses and X-ray diffraction as well. The experimental analyses, useful as a guideline in the restoration, allowed to the knowledge of the original materials as well as of the techniques used to realize the artwork. The paintings are realized by a fresco technique, by using lime and a simple color palette, based on natural earth, as typical of the Byzantine pictorial tradition.