Evidence of a major cosmic event at the end of the Eocene is given by the finding of at least one, possibly two or more, impact horizons containing microtektites, microkrystites, shocked quartz, and unusually high iridium concentrations. We report here the discovery, in the global stratotype of the Eocene-Oligocene boundary at Massignano in Italy, of Ni-rich spinel, a mineral that forms by the interaction of meteoritic bodies with the Earth's atmosphere. A maximum concentration of ≈800 crystals mg−1, corresponding to a flux of 2 × 107 crystals cm−2, is observed in a well-dated layer (35.7 ± 0.4 Ma) that also contains shocked quartz and iridium. The crystals are found clustered in flattened spheroids, the probable relic of their host bodies. The chemical and physical characteristics of the spinel crystals indicate that the spheroids formed by interaction of dust particles in the upper part of the atmosphere rather than by ablation of large objects, as proposed for the spinel-bearing spheroids found at the Cretaceous-Tertiary boundary. The most likely explanation is that the dust particles were generated by a large cometary impact. Whether this impact is the one that produced microtektites and/or microkrystites, or another one that may have occurred shortly after or before, is still questionable. Additional searching for Ni-rich spinel at other upper Eocene sections may help to answer this question.