The High Priestess is the secondary antagonist of the fifth and final season of Samurai Jack. She is the nameless and masked leader of the Cult of Aku as well as the mother and mistress of the Daughters of Aku and lives to serve her god Aku.
She was voiced by Grey DeLisle, who voiced Vicky from the Fairy Odd Parents, Azula from Avatar: The Last Airbender, Asajj Ventress in Genndy Tartakovsky's Star Wars: Clone Wars and Jubileus from Bayonetta.
In "Episode XCII", the High Priestess showed up during a ceremony conducted by the Cult of Aku, an all-female cult of devout worshipers of Aku. There, she gave birth to seven girls and put them through years of hard and intense training to make them into strong ninja assassins who will hopefully track down and kill Samurai Jack, Aku's long-time rival and enemy. After training them, she made them to kill the other cultists and gave them white female masks and weapons and sends them out into the world to kill the samurai.
In "Episode XCV", although the High Priestess didn't appeared physically, Ashi has a dream about her while unconscious, appearing as a nightmarish face demanding her to kill Jack. Later, before she can finish off an exhausted Jack, she sees a ladybug pass and remembers the time her mother squished one for "distracting" her from a sparring session, when Jack holds the ladybug he admires it and let it fly away.
In "Episode XCVI", when Ashi joins Samurai Jack, the High Priestess appeared to Ashi during the night as a vision in the moon telling her to kill Jack while he is sleeping, but Ashi denies to do it explaining to her mother that Jack saved her life and wants to know the truth. The High Priestess angered for Ashi's response then tells her that she has failed her.
In "Episode XCVII", during her quest for Jack who previously went missing with the Omen, Ashi approached a waterfall in the forest and there she remembered the time her mother indoctrinated her and her sisters by having them dive into burning ashes, "becoming one with the darkness" and giving them their signature "outfits". The High Priestess then pushed Ashi first into the pit, showing little care of the screams of pain from her daughter. Diving into the river, Ashi spend the rest of the night scrubbing off her ashes, letting her hair down and creating a new outfit from nearby foliage, finally putting the past behind her.
In "Episode XCVIII", the High Priestess decides to confront the samurai herself along with an army of soldiers while he's distracted with meditating on a mountaintop to retrieve his sword. Ashi manages to defeat all of the soldiers, but the High Priestess sneaks to the top and fires arrows at the samurai, however, Ashi stops them in time. Enraged, the High Priestess battles her final child, accusing her of betraying her deceased sisters by letting the samurai live. However, Ashi denies this and blames the High Priestess for their deaths, by feeding them lies and making their only purpose in life to kill Samurai Jack. When Ashi is dazed by crumbling debris, the High Priestess makes one final attempt to kill the samurai, though when Ashi throws one of her arrows at the High Priestess at the last second, it impales her through the spine and causes her to fall off the mountaintop. It's completely unknown if the High Priestess survived her impalation, but even if she survived that ordeal, she most likely was killed by the fall.
In "Episode C", The High Priestess is shown in a flashback in which she and her cult are visited by Aku. Aku notices the statue they constructed in his likeness and was impressed by the display. As a reward, he gifted them with a small fragment of himself within a chalice, to which the High Priestess drank shortly afterwards. Afterwards, the small bit of Aku caused her to become pregnant with Aku's children, thus giving birth to the Daughters of Aku.
In the series finale "Episode CI", after Jack successfully destroyed Aku in the past and Ashi was erased from existence for helping him to succeed in his mission, the High Priestess eventually never became the leader of the also wiped from existence Cult of Aku, as Aku was never there to have them to appreciate him nor to impregnate her with his essence. However, whether the High Priestess and her daughters (with a human father, of course) still exist in the new timeline or not is unclear, although it could be possible that she instead became a loving mother who loved her children as Aku never had any influence on her life.