Decorated glazed ceramic tiles are used as an ornamental art, constituting an important cultural heritage whose preservation is mandatory. Environmental conditions are responsible for the degradation of exposed ancient tile panels originating various pathologies, related to the development of microorganisms. This is the case of a valuable 18th century blue-and-white Portuguese tile panel called “Cura do Cego,” belonging to the collection of the National Tile Museum (MNAz), where green stains are nowadays observable in the glaze. A prospective diagnosis of this green tarnishing was the aim of the present work. Small tile fragments were directly irradiated using nondestructive techniques: X-ray fluorescence spectrometry with a wavelength-dispersive system (WDXRF) for chemical characterization of the tile glaze and X-ray powder diffraction (XRD) to assess the phase constitution of both the glaze and the ceramic body. A destructive technique (scanning electron microscopy with energy-dispersive system (SEM/EDS)) was applied to tentatively infer the chemical changes induced in the glaze by the green staining and also to characterize the morphology of the microorganisms associated to this staining. The obtained results are reported and discussed, as a preliminary step for testing an innovative nondestructive decontamination technique applying gamma radiation, particularly suitable for overcoming such tile pathologies.