Vergil is sometimes referred to in contemporary documents as Polydore Vergil Castellensis or Castellen, leading some to assume that he was a kinsman of his patron, Cardinal Adriano Castellesi. However, it is more likely that the alias simply indicates that he was in Castellesi's service.
Vergil was born at Urbino, or more probably at Fermignano, within the Duchy of Urbino. His father, Giorgio di Antonio, owned a dispensary. His grandfather, Antonio Virgili, "a man well skilled in medicine and astrology", had taught philosophy at the University of Paris; as did Polydore's own brother, Giovanni-Matteo Virgili, at Ferrara and Padua. Another brother, Girolamo, was a merchant trading with England. The niece of Polydore Vergil, Faustina, married Lorenzo Borgogelli, count of Fano, from whom descend the family of Borgogelli Virgili.
Polydore was educated at the University of Padua, and possibly at Bologna. He was ordained by 1496. He was probably in the service of Guidobaldo da Montefeltro, Duke of Urbino, before 1498, as in the dedication of his Proverbiorum Libellus (April 1498) he styles himself Guido's client. His second book, De Inventoribus Rerum, was dedicated to Guido's tutor, Lodovico Odassio, in August 1499.
At some point prior to 1502 Polydore entered the service of Pope Alexander VI.
In 1502, Vergil travelled to England as the deputy of Cardinal Adriano Castellesi in the office of Collector of Peter's Pence, and, in practice, the Cardinal's agent in a variety of affairs. In October 1504 he was enthroned Bishop of Bath and Wells as proxy for Adriano; and in 1508 he himself was installed as Archdeacon of Wells. He probably spent little time in Wells, but was active as the Chapter's representative in London. He also donated a set of hangings for the quire of Wells Cathedral. He held other ecclesiastical sinecures, including, from 1503, the living of Church Langton, Leicestershire; from 1508 prebends in Lincoln and Hereford Cathedrals; and from 1513 the prebend of Oxgate in St Paul's Cathedral.