We reveal the enigmatic origin of one of the earliest surviving botanical collections. The 16th-century Italian En Tibi herbarium is a large, luxurious book with c. 500 dried plants, made in the Renaissance scholarly circles that developed botany as a distinct discipline. Its Latin inscription, translated as “Here for you a smiling garden of everlasting flowers”, suggests that this herbarium was a gift for a patron of the emerging botanical science. We follow an integrative approach that includes a botanical similarity estimation of the En Tibi with contemporary herbaria (Aldrovandi, Cesalpino, “Cibo”, Merini, Estense) and analysis of the book’s watermark, paper, binding, handwriting, Latin inscription and the morphology and DNA of hairs mounted under specimens. Rejecting the previous origin hypothesis (Ferrara, 1542–1544), we show that the En Tibi was made in Bologna around 1558. We attribute the En Tibi herbarium to Francesco Petrollini, a neglected 16th-century botanist, to whom also belongs, as clarified herein, the controversial “Erbario Cibo” kept in Rome. The En Tibi was probably a work on commission for Petrollini, who provided the plant material for the book. Other people were apparently involved in the compilation and offering of this precious gift to a yet unknown person, possibly the Habsburg Emperor Ferdinand I. The En Tibi herbarium is a Renaissance masterpiece of art and science, representing the quest for truth in herbal medicine and botany. Our multidisciplinary approach can serve as a guideline for deciphering other anonymous herbaria, kept safely “hidden” in treasure rooms of universities, libraries and museums.