In 2019, an exceptional Ptolemaic-era tomb was discovered in Al-Diabat archaeological site, Sohag, Upper Egypt. Numerous fallen painted fragments in the floor of tomb were collected and studied. The microstructural and chemical makeup of samples was discerned by virtue of a field-emission scanning electron microscopy (FE-SEM) with an energy-dispersive x-ray (EDX) spectrometry. Further, the molecularvibrational property was acknowledged by Fourier-transform infrared (FT-IR) and micro-Raman (μ-Raman) spectrometers. Also, the crystalline structure of stone and plaster samples was defined by x-ray diffraction (XRD) analysis. The results have demonstrated that the murals were implemented on a thick mud plaster followed by a thin preparation layer, which, unusually, was made of calcite. Gypsum was also detected in variable quantity throughout the render samples. Colouring substances of Egyptian blue, green earth, red ochre, yellow ochre and carbon black were recognized in the studied murals. Besides, maghemite (γFe 2 O 3) was disclosed in the red pigment sample. Unexpectedly, gold was detected in the yellow paint areas, possibly associated with a religious tradition. As well, the recognition of an organic binder (probably egg yolk) nominates the application of tempera technique. Noteworthy, this research contributes in presenting significant findings on painting materials used in unusual rock-cut tomb from the Ptolemaic era.