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- Akane Yagyū
- Munenori Yagyū - A member of the Yagyū clan, whose heinous acts of betrayal and killing his own kin are the reason Jubei accepts the task to capture her uncle dead or alive. However, though she defeated him, Munenori lived long enough to make a vain assassination attempt on Fortinbras that cost him his life. He is also ironically the person who completes the revival of Fortinbras.
- Roberto - Roberto Frois is a Christian missionary born to a Japanese mother and Spanish father. After being betrayed by Luís Fróis who was under the influence of Rosencrantz, Roberto is now on a mission of pure revenge. But, in a moment of temporary freedom of Rosencrantz control, Luís Fróis places the Exorcising Beads on Roberto's arms, justifying his primary role as Sōki's companion, as this artifact has the power to destroy the Dark Stones. Roberto's weapon of choice are his enhanced arms and fists, capable of numerous destructive boxing combinations. Roberto's gauntlets are based on the Christian archangels, Raphael, Uriel, Gabriel and Michael. In a scene before his battle with Rosencrantz, it is revealed that he is in love with Ohatsu.
- Luís Fróis - Roberto's guardian who betrayed him after being possessed by the Genma high priest/scientist Rosencrantz, one of the three Genma Triumvirate bent on the resurrection of Fortinbras. Once revived, he assumes his true form, a giant fly demon, before slain by Roberto. A moment of clarity after destroying the first dark stone suggests that he truly loved Roberto.
- Minokichi - he, more or less, plays the same role as the Gate Keeper from the original Onimusha.
- Yodo - The daughter of Oyu who became a royal concubine to Hideyoshi. Her true form was a humanoid mantis Genma before she was slain by Ohatsu.
- Sakon Shima - Soki and Tenkai are forced to battle him while he is under the influence of the Genma.
- Ophelia - Ophelia is an insect-like boss, nurturing and caring for the Genma Seed implanted in Hideyoshi's chest.
- Claudius - a boss encountered and destroyed by Tenkai.
- Mitsunari Ishida - Hideyoshi's retainer, whom Claudius possesses. See also Mitsunari Ishida from Sengoku BASARA.
The game takes place some fifteen years after the final defeat of Nobunaga at the end of Onimusha 3: Demon Siege. Nobunaga's vassal, Hideyoshi Toyotomi, takes control of the country, unifying it under one banner and essentially ending the wars that had torn the land apart under his former lord's banner. However, this period of peace ends with several natural disasters acting as an omen of things to come, including the appearance of an "Omen Star" in the sky. Rumors soon fill the land of the Genma returning, and that Hideyoshi might have something to do with it.