The scene depicts the execution of John the Baptist by beheading, with Salome—the daughter of Herod II—stood ready to receive his head on a golden platter. The site of action is undertaken off-centre, with several prisoners emerging from the darkness to observe the execution on the right from their cells.

Widely regarded as one of Caravaggio's most famous paintings, the scene is not directly inspired by the Bible. Instead, it is taken from The Golden Legend, a collection of hagiographies from the Medieval period.

This painting was commissioned by the Knights of Malta (Knights Hospitaller) for use as an altarpiece and drew less on props and models as his earlier career paintings did. It is still available to view at its original place of commission and display, the Saint John's Co-Cathedral in Malta.

Caravaggio even served as a knight for a time, before he committed an unknown crime and again had to flee to another place of residence.

Several other paintings by Caravaggio express the aftermath of this painted scene, the most famous being Salome with the Head of John the Baptist (c. 1607-10), held at the National Gallery, London. The Decapitation of Saint John the Baptist has been damaged over time. In restoring the work in the 1950s, contemporary scholars came across an apparent signature in the painting which is visible in John's blood at the bottom of the painting.