This paper investigates the influence of microorganisms and clay minerals on the deterioration process of ancient Egyptian murals in the Al-Qurna region, situated on the west bank of Thebes (Luxor), Egypt. To achieve this objective, an extensive examination of building materials (including bedrock, building stone, mortars, and plasters) from various tombs within the study area was conducted. The investigation employed a multidisciplinary approach, combining micro-biological analysis with advanced techniques such as X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) as well as petrographic examination. These methods were employed to identify the composition of the materials and to assess the masonry structure’s response to deformation caused by endogenous factors, particularly clay minerals. Additionally, mycological analyses were performed using a swabbing cotton sterile technique, revealing the presence of several species, including Ascosphera apis, Aspergillus tamarii, Aspergillus ochraceus, Doratomyces sp, and Eurotium repens. Notably, swelling clays—such as smectite, illite, and chlorite—along with mixed layers of illite-smectite and illite-vermiculite were detected in the studied samples. The presence of such deleterious clay minerals and fungi imperils wall painting preservation, causing discoloration, surface alteration, physical decay, and structural instability. Informed conservation strategies must address these threats for sustained integrity.