In 1866, the rivalry between Prussia and Austria for supremacy within the German Confederation was at its height. Italy, which wanted to regain Venetia from Austria, concluded a military alliance with Prussia, while Austria formed alliances with the southern German States, which feared the supremacy of Prussia.

War broke out in June 1866. Prussia and Italy had ratified the 1864 Geneva Convention, but Austria and several of its allies had not yet acceded to it. The ICRC tried to remedy the situation, requesting Austria and its allies to be bound by the Convention; most agreed, but Austria refused. The International Committee also requested Prussia and Italy, with some success, to apply the Geneva Convention unilaterally.

Lastly, the Committee facilitated communication among the belligerent countries' relief societies, and between them and the societies of neutral countries. On the other hand, contrary to what it had done during the German-Danish War, it did not act directly to assist the victims of the conflict.

The war ended with the Prussian victory at Sadowa. Austria acceded to the Geneva Convention three weeks later, on 21 July 1866.