The application of non-invasive geophysical techniques and digital surveys to explore cultural heritage is becoming a very important research field. The capability to detect inner and superficial changes in the inspected surfaces allows for imaging spatial inhomogeneity and material features and planning targeted conservation and restoration interventions. In this work, the results of a research project carried out on the famous Battle of Issus Mosaic, also known as the “Alexander Mosaic”, are presented. It is a masterpiece of ancient art that was found in 1831 in the House of Faun, the most luxurious and spacious house in Pompeii. It is notable for its size (3.41 × 5.82 m), the quality of workmanship and the subject that represents the culminating phase of the battle between Alexander Magno’s army and the Persian one of Darius. In 1916, it was moved inside the National Archaeological Museum of Naples, where the original horizontal location was changed with a vertical arrangement supported by an inner wooden structure, whose exact manufacture is unclear. Today, the mosaic is affected by important instability phenomena highlighted by the appearance of the significant detachment of tiles, superficial lesions and swelling of the surface. Given the important need to preserve it, a high-detail diagnostic study was realized through a digital survey and non-invasive geophysical surveys using ground-penetrating radar (GPR). The investigation was repeated after two years, in 2018 and 2020, with the aim of verifying the evolution of degradation. The work provided a high-resolution estimate of the state of the health of the mosaic and allowed for obtaining a three-dimensional reconstruction of the internal mosaic structure, including the formulation of hypotheses on the engineering supporting works of the twentieth century; this provides an essential tool for the imminent conservation project, which also implies restoring the original horizontal position.