Abstract The crypt of St. Nicholas in Bari, Southern Italy, is a building of cultural worldwide importance. Inside the crypt a mosaic develops on the apsidal floor and along a parietal seat placed along the apsidal masonry, in the form of cladding. A surviving portion of the mosaic pavement is also preserved in the right lateral chapel. Integrated non-destructive survey and laboratory analyses were undertaken for the diagnostic study of the mosaic. GPR prospection was successfully applied to the study of its conservation state, that is strictly related to the condition of the subsoil, in particular to the presence and distribution of water. The presence of water, whose rise interests also some portions of the masonry and water content distribution were identified by the results of electromagnetic wave velocity analysis in GPR data. The decay visible on the tesserae evidenced widespread spalling and crumbling as typical forms that selectively affect the different materials of the tesserae of the mosaic. Samples taken from them were investigated by optical microscopy, X-ray diffraction, ion chromatography, infrared spectroscopy and thermogravimetric analyses, in order to identify the constituent materials and the products of their decay. The application of the integrated methodologies showed its effectiveness in order to acquire a quite complete knowledge for diagnostic purpose. Decay appeared to be due to the combined presence of water and soluble salts and it has different effects depending on the materials characteristics.