The Doni Tondo portrays the Holy Family (the child Jesus, Mary, and Joseph) in the foreground, along with John the Baptist in the middle-ground, and contains five nude male figures in the background.

The scene appears to be a rural one, with the Holy Family enjoying themselves on the grass and separated from the curiously (seemingly) unrelated group at the back by a low wall.

Because they are much closer to us, the viewers, the Holy Family is much larger than the nudes in the background, a device to aid the illusion of deep space in a two-dimensional image.

This semi-circle reflects or mirrors the circular shape of the painting itself and acts as a foil to the vertical nature of the principal group (the Holy family).

The Doni Tondo is believed to be the only existing panel picture Michelangelo painted without the aid of assistants;[7] and, unlike his Manchester Madonna and Entombment (both National Gallery, London), the attribution to him has never been questioned.

The composition is, most likely, partially influenced by the cartoon (a term referring to a detailed later-stage preliminary drawing) for Leonardo da Vinci's The Virgin and Child with St. Anne.

Michelangelo's Holy Family forms a tight, separated group in the centre foreground of the image, with the Virgin's figure constructing a typical Renaissance pyramid or triangle.

Signorelli's Madonna similarly uses a tondo form, depicts nude male figures in the background, and displays the Virgin sitting directly on the earth.

[6] Three aspects of the painting can be attributed to an antique sardonyx cameo and a 15th-century relief from the circle of Donatello, available to Michelangelo in the Palazzo Medici: the circular form, the masculinity of Mary, and the positioning of the Christ Child.

Furthermore, the inclusion of the five protruding heads in the paintings frame is often seen as a reference to a similar motif found on Ghiberti's Porta del Paradiso, the bronze doors of the Florence Baptistry which Michelangelo is known to have greatly admired.

[21] Barolsky bases much of his thesis on the language used by Giorgio Vasari in his work Lives of the Most Excellent Italian Painters, Sculptors, and Architects, from Cimabue to Our Times.

[22] Mirella Levi D'Ancona argues that the image reflects Michelangelo's views on the roles of the members of the Holy Family in human salvation and the soul's immortality.

[11] Andrée Hayum argues that the commissioning of the tondo by the Doni family helped to emphasize the "secular and domestic ideals" of the painting rather than seeing it as a "devotional object".

[3] In choosing a tondo as the format for the picture, Michelangelo is referencing the form's long association with depicting the "Adoration of the Magi, the Nativity, [and] the Madonna and Child.

[24] Hayum further supports this by acknowledging the direct link between Joseph and Noah as depicted in Michelangelo's Sistine Ceiling paintings.

[28] Much importance is given to Joseph by way of the colors of his clothes: yellow, indicating the divine aspect of the family as well as "truth", and purple, standing for royal lineage tracing from the House of David.

[29] The theme of baptism is also suggested on the painting's frame through a possible reference to Ghiberti's Porta del Paradiso – being one of the three sets of doors of the Florentine Baptistry (two of which by Ghiberti) – the sculpted details indirectly referring to the rite of Baptism, important for the Donis and their desire for a child as the product of a good marriage, exemplified by the Holy Family, perhaps one reason behind the commissioning of the work.