Ayer manuscript 1478, held by the Newberry Library in Chicago, is a fascinating document offering us a unique insight into the cultural and linguistic contact between the Nahua and the European worlds that occurred in sixteenth-century New Spain. This handwritten copy of the Spanish-Latin dictionary of E.A. de Nebrija supplemented with glosses in Nahuatl is probably the oldest extant lexicographic work on an indigenous American language. The study, created within the ERC project “Europe and America in contact: a multidisciplinary study of cross-cultural transfer in the New World across time,” analyzes the Nahuatl lexis attested in the manuscript and focuses on how it was influenced by European culture and languages.