[3][4] The meaning of the two other male figures on the left side (the Madonna's right) is, like the overall iconography of the image, not entirely clear.

Franny Moyle writes that the man on the Madonna's right is Meyer, and "[t]he boy and baby may be a presentation of Meyer's two deceased sons...."[5] The image testified to the resolutely Catholic faith of the Bürgermeister, who actively opposed the Reformation.

Holbein's Darmstadt Madonna was influenced by Italian Renaissance religious painting; Franny Moyle writes that it was influenced by Andrea Mantegna's 1496 Madonna della Vittoria, and that the two paintings' "perspectival approach ... is almost identical.

Earlier located in Darmstadt, hence its title, the work was on loan to the Städelschen Kunstinstitut in Frankfurt am Main from 2004 to 2011.

In 2012, the painting was put on display in the Johanniterkirche in Schwäbisch Hall as part of the permanent exhibition of the Old Masters in the Würth Collection.