"Isekai Quartet" is a crossover anime that brings together characters from four popular isekai series: "Re:Zero - Starting Life in Another World," "Overlord," "KonoSuba: God's Blessing on This Wonderful World!," and "The Saga of Tanya the Evil." The narrative begins when a mysterious button appears in each of the respective worlds of these characters. Upon pressing it, they are transported to a new, unfamiliar world where they find themselves enrolled in a school-like setting. The story unfolds as they navigate this strange environment, interact with one another, and adapt to their new circumstances.

The main characters include Subaru Natsuki, Emilia, and Beatrice from "Re:Zero"; Ainz Ooal Gown, Albedo, and Shalltear from "Overlord"; Kazuma Satou, Aqua, Megumin, and Darkness from "KonoSuba"; and Tanya Degurechaff and Visha from "The Saga of Tanya the Evil." Each character retains their unique abilities, personalities, and quirks, which often lead to humorous and chaotic situations as they clash or collaborate. The series explores their daily lives in this new world, including attending classes, participating in school events, and dealing with the challenges posed by their vastly different backgrounds and ideologies.

The plot is episodic, with each episode focusing on various comedic scenarios and interactions between the characters. The humor often stems from the contrast between their serious, high-stakes original worlds and the lighthearted, school-based setting of "Isekai Quartet." Despite the comedic tone, the series occasionally touches on the characters' underlying motivations and relationships, providing moments of depth and continuity with their original stories. The narrative does not follow a traditional overarching plot but instead relies on the charm and chemistry of the ensemble cast to drive the series forward.