The initial nucleus of gemstones at the Natural History Museum of the University of Florence (Italy) is linked to the significant collection of the Medici family, who began it as early as the 15th century. The present research aims to study this collection in order to (1) comprehensively review the archival and catalogue information available; (2) identify the mineralogical species correctly; and (3) gather information on the potential provenance of the gem deposits. To address these objectives, fifty gems were investigated using entirely non-invasive methods, ensuring the preservation of the collection’s precious and historical value. All specimens underwent autoptic observation and micro-Raman analysis, while a selection was further examined using PIXE-PIGE to characterise their chemical composition, including trace elements. The gems were attributed to seven mineral species: emerald, topaz, grossular, cordierite, quartz, orthoclase, and tourmaline. One gem was identified as a fake, made of glass and likely produced in the 17th century. Twenty-nine of the historical attributions in the catalogue were found to be incorrect and were subsequently revised. In some cases, the trace elements and mineral inclusions identified in the gems enabled the determination of potential provenance deposits, which were then compared with the available archival information.