Mithraea, religious Roman buildings, are very common in Italian archeological sites. There are sixteen in Ostia Antica (Rome, Italy)The poor state of conservation, due to the intrinsic environmental conditions, characterized them: they consist of open-air museums and caves simultaneously. These places of worship are characterized by the presence of heterogeneous materials, such as wall building materials (bricks and mortars) and others used for furnishings and fittings. This increases the risk of accelerated damage because the materials ‘rheology is different. Here, a full petrographic-mineralogical characterization with polarized light microscopy (PLM), X-ray diffraction (XRD), scanning electron microscope with energy dispersive X-ray (SEM-EDS) and isotopic analysis (δ13C, δ18O) is carried out on materials like travertine, marble, pumice, ceramic, and wall-building materials in “Casa di Diana” Mithraeum (Ostia Antica). Their characterization gives provenance information as well as conservation and restoration purposes. The prevalence of siliciclastic or carbonate components discriminates between red and yellow bricks, as well as different textures and minerals in the aggregate of the red ones. The mortars are typically pozzolanic, and the aggregate is mostly made up of black and red pozzolanic clasts. In the altar, apse, and aedicule, which constitute the principal place of the Mithraeum, a variety of materials used for the ornamental purpose are represented by pumices, travertine, marble, and limestone. The altar material, catalogued as marble, resulted in being a limestone coated with a white pigment.