Mitsunari was the son of Ishida Masatsugu and was born at Ishida in Ômi Province. He was recruited into Hashiba (Toyotomi) Hideyoshi's service around 1578, in part due to his cultural acuity. While he saw military service at Shizugatake and elsewhere, his main function was that of an administrator. He accumulated a fief of some 200,000 koku and was given Sawayama Castle in Ômi. He became distrusted and disliked by many, in part due to his 'civilian' nature and in part to the power he wielded within the Toyotomi goverment. He issued numerous orders in Hideyoshi's name and often acted as Hideyoshi's representative. He was dispatched to Korea during the second campaign there in 1597 as Inspector of Forces. In the course of carrying out his duties he earned the hatred of both Kuroda Yoshitaka and Kobayakawa Hideaki, whom ishida reported as being lax in their conduct.

In 1598 Mitsunari was named one of the Five Commisoners (san-bugyo) responsible with maintaining the civil affairs of the realm while Hideyori came of age. He was out-spoken and at times tactless, but held enough support to challenge Tokugawa Ieyasu, the most powerful of the Regents. He argued - with some cause - that Ieyasu was undermining both the legacy of the late Taikô and his final wishes. Ieyasu countered by painting Mistunari (also with some validity) as an unscrupulous schemer. Mistunari went so far as to attempt the assassination of Ieyasu in 1599, and narrowly avoided his own death at the hands of several Tokugawa loyalists (thanks, ironically and mysteriously, to help from Ieyasu himself).

The following year, after gaining the support of three of the Regents (Môri Terumoto, Uesugi Kagekatsu, and Ukita Hideie), Mitsunari rallied a host of daimyô (predominantly from the western provinces) against Ieyasu. One of his first acts was to take as hostages the wives of Tokugawa supporters who happened to be in Osaka. On 22 August the Sekigahara Campaign began. In the lead-up to the climactic battle, Mitsunari argued with Môri Terumoto and named the half-hearted lord of the Môri nominal commander of the 'western' forces. Frustrated by Terumoto's reluctance, Mitsunari asked him to guard Hideyori at Osaka Castle. This evident ploy on Mitsunari's part to maintain his importance in the unfolding events deeply insulted the Môri, and in the Battle of Sekigahara on 21 October that clan would contribute little. In the meantime, Mitsunari and his compatriots had hoped that Uesugi Kagekatsu would be able to delay Ieyasu himself from marching west long enough for the Western forces to consolidate their hold on the provinces around Kyoto. To this end, a number of Western contingents became involved in sieges that would only serve to remove them from the decisive field of battle. To Western dismay, Kagekatsu was rather handily contained by Date Masamune and Mogami Yoshiaki and Ieyasu moved quickly westward on the Tokaido road. Gifu Castle, in Mino Province, held by Western ally Oda Hidenobu, fell to an attack by Fukushima Masamori and Ikeda Terumasa, clearing the way for Tokugawa's main body. Yet the field remained more or less even between the two main armies, as another Tokugawa army, led by Ieyasu's heir, Hidetada, became pointlessly wrapped up in an abortive attack on Sanada Masayuki's Ueda Castle in Shinano Province.
The sudden movement of Ieyasu saw a valley surrounding the village of Sekigahara nominated by default as the battleground. Mitsunari's make-shift strategy was sound - he intended to draw Ieyasu into the valley and fall on him from all sides. To this end Ukita, Konishi, Otani, Shimazu, and others were positioned to the eastern edge of the valley, while, on high ground to the south, Kobayakawa's large contingent was drawn up, reinforced there by the Wakizaka and others. The Chosokabe, Môri, and Ankokuji deployed on the hills to the south east of the rest of the army and were destined to contribute almost nothing to the contest, owing in large part to Môri's disinclination to offer battle. Yet, the Western forces were in the actual event undone by the betrayal of Kobayakawa and those positioned with him. Mitsunari's coalition, shaky and of uneven quality even prior to the battle, was utterly defeated, and Mistunari himself was apprehended some days later. In the meantime, the Ishida's castle of Sawayama was attacked and Mitsunari's brother, Masazumi, commited suicide. Mitsunari's father followed suit. Mitsunari himself was taken to the Rokujôgahara execution grounds in Kyoto and was beheaded along with Ankokuji Ekei and Konishi Yukinaga.