The present paper illustrates the results of a diagnostic investigation performed on the oil on canvas painting “The Glory of St. Barbara” (1680–1688) by Mattia Preti. The painting is located inside the St. Barbara Church in Taverna (Catanzaro, Italy), the city that gave birth to the artist. In situ, non-invasive studies, by applying X-ray fluorescence (XRF) spectroscopy, along with laboratory micro-destructive analytical investigations, by employing electron probe microanalyses (EPMA) coupled with energy-dispersive spectrometry (EDS) and micro-Raman (µ-Raman) spectroscopy, were combined to retrieve the color palette and identify the painting technique and style of the famous master. As a result of this multi-scale characterization, an extraordinary pictorial technique was revealed, enriching knowledge about one of the oldest pictorial traditions outlined by Mattia Preti, and solving doubts still existing about many of his investigated artworks. Moreover, the achieved results represent useful and essential tools to address management issues of the artwork, by providing valuable information for planning and monitoring future restoration interventions of the canvas.