Ishii led the development and application of biological weapons at Unit 731 in China during the Second Sino-Japanese War from 1937 to 1945, including the bubonic plague attacks at Chinese cities of Changde and Ningbo, and planned the Operation Cherry Blossoms at Night biological attack against the United States.

Ishii and his colleagues also engaged in human experimentation, resulting in the deaths of over 10,000 subjects, most of them civilians or prisoners of war.

The Ishii family was the community's largest landholder and exercised a feudal dominance over the local village and surrounding hamlets.

His daughter Harumi felt that Shiro had been "unjustly condemned", saying "my father was a very warm-hearted person...he was so bright that people sometimes could not catch up with the speed of his thinking and that made him irritated, and he shouted at them.

Ishii's travels were highly successful and helped win him the patronage of Sadao Araki, the Japanese Minister of the Army.

[9] As the leader of Unit 731, Ishii conducted a variety of experiments, including vivisections,[10] testing biological weapons on Chinese villages,[11] poisoning by toxins and gases[12][13] and forcing inmates to inflict syphilis on each other.

[16] Towards the end of the war, Ishii would develop a plan to spread plague fleas along the populated west coast of the US, known as Operation Cherry Blossoms at Night.

[18] Ishii was later granted immunity in the International Military Tribunal for the Far East by the United States government in exchange for information and research for the U.S. biological warfare program.

[19] Instead, Ishii and his team managed to negotiate and receive immunity in 1946 from Japanese war crimes prosecution before the Tokyo tribunal in exchange for their full disclosure.

After being granted immunity, Ishii was hired by the U.S. government to lecture American officers at Fort Detrick on the uses of bioweapons and the findings made by Unit 731.

[23][24] During the Korean War, Ishii reportedly traveled to Korea to take part in the U.S. Army's alleged biological warfare activities.

[25] On 22 February 1952, Ishii was explicitly named in a statement made by North Korean Foreign Minister Pak Hon-yong, claiming that he, along with other "Japanese bacteriological war criminals",[26] had been involved in "systematically spreading large quantities of bacteria-carrying insects by aircraft in order to disseminate contagious diseases over our frontline positions and our rear".

[26] However, whether the U.S. Army actually used biological weapons against Chinese or North Korean forces, or whether such allegations were mere propaganda, is disputed by historians.

Shortly before his death, he asked to be baptised by the late Dr Herman Heuvers, former President of Sophia University in Tokyo.