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This study presents the petrographic and chemical characterization of the frescoes in the Church of San Panfilo in Tornimparte (AQ, Italy) by Saturnino Gatti, a prominent painter of the late 15th–early 16th century, known for his exquisite technique, composition, and use of color. The characterization of the frescoes is essential for understanding the materials and techniques used by Gatti, as well as for identifying the stratigraphy and painting phases. Eighteen samples were collected from the original paint layers, later additions (17th century), and restored surfaces, and analyzed by optical microscopy, cathodoluminescence microscopy, scanning electron microscopy (SEM-EDS), μ-Raman, and electron paramagnetic resonance (EPR). The analyses revealed a microstratigraphy often made of three main layers: (1) preparation, consisting of lime plaster and sand; (2) pigmented lime, applied by the fresco technique; and (3) additional pigmented layer on the surface. The most often recurring pigments are black, red, yellow (all generally linked with the fresco technique), and blue (applied “a secco”). The presence of two painting phases was also noted in one sample, probably resulting from a rethinking or restoration. These findings contribute to the understanding of the history and past restoration works of this cultural heritage site, providing important insights not only for conservators and restorers, but also for a broader understanding of Italian fresco painting and art history of the late 15th and early 16th centuries.