"Hyakka Ryōran Samurai Girls" is set in an alternate version of Japan, where the country is ruled by the Tokugawa shogunate in a modern era. The story unfolds at Buou Academic School, a prestigious institution where students train to become samurai. The protagonist, Muneakira Yagyu, is a second-year student and the last remaining practitioner of the Yagyu sword style. His life takes a dramatic turn when he encounters Jubei Yagyu, a mysterious girl who falls from the sky and claims to be a master samurai. Jubei possesses the ability to awaken the latent powers of other girls, transforming them into "Master Samurai" through a kiss.

As the narrative progresses, Muneakira becomes entangled in a conflict involving the Tokugawa shogunate and a secretive organization known as the Anti-Tokugawa forces. The shogunate seeks to maintain control over Japan by suppressing any threats to its authority, while the Anti-Tokugawa forces aim to overthrow the regime. Muneakira and Jubei are joined by other female warriors, each with their own unique abilities and ties to historical samurai clans. These include Yukimura Sanada, a fiery and determined fighter; Sen Tokugawa, the shogun's granddaughter with a calm and strategic demeanor; and Gisen Yagyu, a rival samurai with a complex relationship to Muneakira.

The series blends historical elements with fantasy, as the Master Samurai wield supernatural powers tied to their ancestral lineages. The characters navigate political intrigue, personal rivalries, and battles against powerful enemies, all while uncovering the truth behind Jubei's origins and the shogunate's hidden agenda. Themes of loyalty, honor, and the clash between tradition and modernity are explored as the characters grapple with their roles in shaping Japan's future. The story balances action, humor, and moments of introspection, creating a dynamic narrative that appeals to fans of historical fantasy and samurai lore.