The Legendary Chalice can be found inside three mausoleums within the Inkwell Isles, one in each respective section. Inside, she will call out for help from the brothers inside an urn, which the brothers must defend from hostile ghosts.

The ghosts will not harm the brothers, but they can only be defeated by parrying them, as indicated by their pink colors. Each mausoleum introduces different ghosts that have different movement patterns or require multiple parries in order to defeat them. Once all the ghosts have been defeated, the Legendary Chalice will emerge from the urn. She will thank the brothers for their help and grant them one special ability known as a "Super Art".

She can be summoned by using the Astral Cookie Charm to replace the player's current character. Ms. Chalice begins each stage with a default of four hit points instead of three and can use unique abilities that the brother's cannot perform themselves, such as a double jump and a evasive roll. Her attacks during aeroplane stages also work differently.

In the story, the Legendary Chalice meets with Cuphead and Mugman on Inkwell Isle 4 to tell them that she had amazing news to share and it all had to do with a special cookie she was holding. She has Mugman take a bite of a cookie which suddenly causes him to become a ghost and brings Chalice back to life. Thrilled that she was alive again, Chalice leads both boys to the one who made the cookie while explaining that, until just now, she had tried everything to leave the astral plane with no success. They enter a bakery where Chalice introduces them to Chef Saltbaker, the finest baker in all the lands, before she and Mugman immediately revert to their previous states. Saltbaker remarks that the cookie's effects were only a temporary solution to Chalice's predicament and tells the trio that he can make a pastry called the Wondertart that can give them control over the astral plane, which would restore Chalice to life without the need to switch forms with someone else. But first, they must gather each of the five ingredients that are being guarded somewhere on the island.

After the heroes obtain all the ingredients they needed to make the Wondertart, Chef Saltbaker reveals that he had no intention on helping Chalice and plans to sacrifice one of their souls to complete the pastry and take control of the astral plane himself. However, the heroes foil Saltbaker's plot and destroy his bakery before Chalice decides to find another way to revive herself. She then embraces Cuphead and Mugman, reminding them that they can still use the cookie she gave them to call for her help anytime.