The study concerned a diagnostic spectroscopic campaign carried out on the panel painting depicting the Coronation of the Virgin (first half of the 15th century) by the late-Gothic Italian painter Michele di Matteo. The main aims were the identification of the original painting materials and the characterization of the painter’s artistic technique. A combined approach based on non- and micro-invasive techniques was employed. Visible and ultraviolet-induced fluorescence photography was used to select the areas of interest for spectroscopic analyses; X-ray radiography assessed the state of conservation of the support, while X-ray fluorescence and external reflection Fourier transform infrared spectroscopies allowed the chemical identification of pigments, binders, and varnishes. Attenuated total reflection infrared spectroscopy, optical microscopy, and scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy were used to visualize and characterize the materials in the pictorial layers. The results highlighted the presence of pigments, possibly applied with an egg binder, consistent with the period of the production of the painting, as well as modern pigments used during subsequent restorations: an imprimitura with lead white and a gypsum-based ground layer. Concerning the gilding, the guazzo technique was confirmed by identifying a red bolo substrate and gold leaf.