The traditional account of his life was based on the biography written by the 16th century Flemish artist and art historian Karel van Mander and included in his Schilder-boeck, published in Haarlem in 1604.

Recent art historians think he probably worked in the provincial town of Haarlem, some way from the larger cities of the southern Netherlands, for all or most of his career.

In 1500 Mostaert was commissioned to paint the shutters for a receptacle housing the relics of Saint Bavo in the Groote Kerk, Haarlem.

[2] Margaret of Austria, the governor of the Habsburg Netherlands, appointed Mostaert 'painctre aux honneurs' (painter with honors) in March 1518.

From Geertgen, Mostaert adopted a refined style and thoughtful compositions for his works, as well as the stiff, angular look of his figures.

Between 1510 and 1516, Mostaert developed a delicate style where his doll-like figures inhabited bright, blue-skied landscapes, as for example in his Adoration of the Magi (c. 1520–25, Rijksmuseum).

Mostaert's St. Christopher, a painting with a landscape that features a river receding into an expansive and hilly background, was once even attributed to Patinir.

As was common practice in 15th and 16th century Netherlands, Mostaert frequently reproduced portraits of political figures based on original models.

It is not known exactly who this man was but there are indications that he was either associated with Margaret's court or was an attendant of her nephew, Charles V, who had a black archer, Christophle le More, among his bodyguards.

There was speculation that the artist may have seen sketches of the landscapes but this is unlikely since most pictorial documentation of the times was of exotic animals, tools or costumes, rather than panoramic views.

It is believed that Mostaert created the painting based on either written or oral accounts of the newly discovered area to which he would have been privy thanks to his contacts at the court of Margaret of Austria, the aunt of Charles V. Although the narrative in the painting was thought to depict a number of different events, including Columbus on the island of Guanaja, Hernán Cortés in Mexico, and the Portuguese invasion of Brazil, it is actually the story of Coronado's search for the Seven Cities of Gold in the Zuni village of Cibola in New Mexico and Arizona in 1540–42.

The landscape's terrain, as well as the look of the natives, also matches the descriptions of Pedro de Castañeda, a member of Coronado's team, made during his travels.

Mostaert devotes only a small section of the painting to Coronado and his men, showing us instead the unity and strength of the native peoples fighting for their land.

Mostaert's Portrait of a Courtier, a painting that until 2005 had been in the collection of the Virginia Museum of Fine Arts since 1949, was found to have been confiscated by Nazi forces in 1941 from its rightful owners in Poland.

The VMFA made the decision to deaccession and transfer the work to the Polish Embassy, acting on behalf of the rightful owners' family.