This current article is focused on the characterization of the preparatory mortar layers and tesserae from the mosaic floor of the Hippolytus Hall at Madaba, located in southwest Jordan. The chemical analysis of the ancient mosaic mortar and tesserae can provide valuable data regarding the an-cient materials and technological processes applied for the production of the mosaic floor in the Byzantine period in Jordan. As well as, the conservation of the mosaic requires knowledge concerning the construction techniques, properties of the used materials as well as the deterioration factors. For these purposes, a consid-erable group of stone tesserae of different colors were collected together with two mortar samples from the preparatory layers and analyzed by X-Ray Diffraction (XRD) and Scanning Electronic Microscope attached to energy dispersive X-ray spectrometer (SEM-EDX). The results showed that the mosaic floor composed of three layers (statumen, nucleus, and supra nucleus). The supra nucleus and nucleus layers mortars are a lime-based mortar and the Calcite (CaCO3) is the main mineral of both samples, which indicates that the mortar used in preparatory layers of the mosaic is localized lime mortar. In addition, the good adhesion between mosaic layers can explain the favourable state of conser-vation, stability and long-term durability of this mosaic floor Besides, these results revealed that Calcite is the main mineral of all limestone tesserae samples (CaCO3). The calcite here is a microcrystalline Calcite is known as Micrite.