The obsidian mirror associated with the Elizabethan polymath and magus John Dee (1527–1608/1609) has been an object of fascination for centuries. The mirror, however, has a deeper history as an Aztec artefact brought to Europe soon after the Spanish conquest. The authors present the results of new geochemical analysis, and explore its history and changing cultural context to provide insights into its meaning during a period in which entirely new world views were emerging. The biography of the mirror demonstrates how a complex cultural history underpins an iconic object. The study highlights the value of new compositional analyses of museum objects for the reinterpretation of historically significant material culture.