Holbein painted this work in the early part of his second stay in England, which began in 1532. At this time he was prolific, painting Hanseatic merchants, courtiers, landowners, and visitors. The Ambassadors is his most famous and perhaps greatest painting of this period. The life-sized panel portrays Jean de Dinteville, an ambassador of Francis I of France in 1533, and Georges de Selve, Bishop of Lavaur, who visited London the same year.

Until the publication of Mary F. S. Hervey's Holbein's Ambassadors: The Picture and the Men in 1900, the identity of the two figures in the picture had been a matter of intense debate. In 1890, Sidney Colvin was the first to propose the figure on the left as Jean de Dinteville, Seigneur of Polisy (1504–1555), French ambassador to the court of Henry VIII for most of 1533. Shortly afterwards, the cleaning of the picture revealed his seat of Polisy as one of only four places marked on the globe. Hervey identified the man on the right as Georges de Selve (1508/09–1541), Bishop of Lavaur, after tracing the painting's history back to a 17th-century manuscript. Hervey's identification of the sitters has remained the standard one, affirmed in several extended studies of the painting. However, rival speculation is not entirely dead. Opposition to the identification of de Selve has been based on an inventory of 1589, discovered by Riccardo Famiglietti, which names the man on the right as de Dinteville's brother François. Leading scholars of the painting argue that this identification of 1589 was incorrect.