The Ark of the Covenant was a Hand of God, an item touched by God and containing a portion of his power.
In Caged Heat, Sam claimed to have found the Ark of the Covenant in order to get the attention of Castiel. After Castiel showed up, Sam told him that he'd lied and been using the plot of Indiana Jones and the Raiders of the Lost Ark to get Castiel's attention.
In 1943, Nazi archeologists located a fragment of the Ark of the Covenant and aware that it was a Hand of God, saw it as a way to win World War II. The Men of Letters sent Delphine Seydoux to steal the Ark and bring it back to America to be stored in the Men of Letters bunker for safekeeping. Delphine killed Gestapo leader and Thule Gumprecht and stole the Ark and began transporting it to America on the USS Bluefin, an American submarine.
Having learned of the Ark in 2016, Dean Winchester had what he believed to be Castiel but was in fact Lucifer transport him back in time to the Bluefin in hopes of using the Ark as a weapon against the Darkness. The Bluefin came under attack from a Nazi destroyer commanded by a revived Gumprecht and Delphine decided to use the power of the Ark to stop Gumprecht and his crew from getting their hands on it. Delphine absorbed the power of the Hand of God from the Ark and used it to unleash a blast that obliterated the Bluefin and burned a hole through the destroyer, sinking it and killing Gumprecht and his crew.
Before the Bluefin was obliterated, Lucifer transported Dean and the Ark fragment back to the bunker in 2016. There, Lucifer attempted to use the power of the Ark to kill the Winchesters before going after the Darkness, but found that Delphine's use had used up the Ark's power. Lucifer's distraction over the Ark piece enabled Sam to banish Lucifer using an angel banishing sigil and Lucifer left the Ark behind. As Dean contemplated the Ark fragment, Sam informed him of what happened when Delphine used the Ark's power against the Nazis.
- In the lore, The Ark of the Covenant (Hebrew: אָרוֹן הַבְּרִית ʾĀrôn Habbərît, modern pron. Aron haBrit), also known as the Ark of the Testimony, was a wooden chest clad with gold containing the two stone tablets of the Ten Commandments as well as, according to various texts within the Hebrew Bible, Aaron's rod and a pot of manna. In lore, it was often a means used by people to communicate with God. According to the lore, The Ark has been lost since the Babylonian conquest of Jerusalem in 587 BC.
- In the Old Testament, the Ark was immensely powerful and touching it was forbidden. While being carried by mules, one of the mules collapsed causing the Ark to fall. A man named Uzzah caught the Ark before it could hit the ground. But touching it earned God's Wrath and Uzzah was smited.
- The Ark was made most famous by the movie Raiders of the Lost Ark, which Sam refers to when luring Castiel to him.