E.I. du Pont de Nemours & Company was organized in Paris in 1801 by Pierre Samuel du Pont de Nemours (1739-1817) and his son Eleuthère Irénée "E.I." du Pont (1771-1834). The next year, E.I. du Pont purchased a mill site on the banks of the Brandywine River just north of Wilmington, Delaware, and began making preparations to establish a black powder manufactory. During the winter of 1802 to 1803, E.I. du Pont recruited a small workforce and began building the company's mills. By the spring of 1804, the powder was being produced, and the company had secured a commitment from President Thomas Jefferson to purchase DuPont gunpowder for the Army and Navy. With this contract in place, the company started to recruit immigrant Irish workers for the mills and hired its first independent sales agents.

During the War of 1812, DuPont became a major supplier of gunpowder for the U.S. government as its total sales exceeded 500,000 pounds. After the war, the company expanded as it began selling large quantities of powder to coal mine operators and railroad entrepreneurs. In 1837 Henry du Pont (1812-1889), E.I. du Pont's son, took over the management of the company and began to rationalize the company's managerial practices. During the Civil War, DuPont became the largest supplier of powder for the Union Army, and in the late 1860s and 1870s, it used its wartime profits to purchase control of many of its competitors.

In 1872 DuPont organized the Gunpowder Trade Association, which acted as a cartel to limit competition and raise prices. In the late 1880s and early 1890s, the company began experimenting with smokeless powder, and it purchased the rights to distribute dynamite in America from Alfred Nobel. By the late nineteenth century, the company was beginning to lose market share as its mills were becoming obsolete. In 1899 DuPont incorporated and began to modernize its mills and rationalize its archaic management structure.

Throughout the 1900s and 1910s, the company shifted its focus away from gunpowder production and towards chemistry innovations.