The decree at the Catholic Council of Trent confirming their canonical status was passed in April 1546, around the time this painting is thought to have been made, which may have a bearing on the choice of subject.

The painting shows the moment from Chapter 9:6–8, when Esther goes to see her husband, King Ahasuerus of Persia (often identified as the historical Xerxes I), to intercede for the Jewish people; going into the inner royal court uninvited is punishable by death:[5] 6 Then having passed through all the doors, she stood before the king, who sat upon his royal throne, and was clothed with all his robes of majesty, all glittering with gold and precious stones; and he was very dreadful.

7 Then lifting up his countenance that shone with majesty, he looked very fiercely upon her: and the queen fell down, and was pale, and fainted, and bowed herself upon the head of the maid that went before her.

[8] The fainting became a much more popular subject in the Baroque painting of the following century, with examples including the Esther Before Ahasuerus by Artemisia Gentileschi.

What appears now probably represents Tintoretto's initial intention, but in the course of painting he abandoned the two figures standing behind the king, a tall twisting one with a long scarf who was presumably Haman, and what is now a very dim outline of a turbaned companion.

The Escorial version also extends the composition to both sides, and very slightly at the top and bottom, adding one and a half figures at the right, and showing the full leg and foot of the boy with a dog at left.