In 1801, French immigrants Eleuthère Irénée "E.I." du Pont (1771-1834) and his father Pierre Samuel "P.S." du Pont de Nemours (1739-1817) organized the E.I. du Pont de Nemours & Company (alternatively referred to as the DuPont Company) in Paris for the purpose of manufacturing gunpowder in the United States. The next year E.I. du Pont purchased a mill site on the banks of the Brandywine Creek just north of Wilmington, Delaware, and began preparing to establish a black powder manufactory. During the winter of 1802-1803, E.I. du Pont recruited a small workforce and began building the company's powder yard, which he named Eleutherian Mills. By the spring of 1804, the powder was being produced, and the company had secured a commitment from President Thomas Jefferson (1743-1826) to purchase 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 to sell and market powder in their home locations throughout the United States.

By 1811, the du Ponts' mills had already become the largest in the United States for the manufacture of gunpowder. During the War of 1812, the company 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 1813, an additional 62 acres were purchased adjacent to the original yard for another powder yard, which was named Hagley Yard. The company's early years were not without struggle, however. P.S. du Pont de Nemours passed away in 1817 after helping to control a fire that had broken out in the yards. The company suffered another setback with the "Great Explosion" on March 19, 1818, in the Eleutherian Mills yard, which killed 34 people. In a series of three explosions, the Glazing Mill, Packing House, and Powder Magazine were destroyed by over 50,000 pounds of black powder.

E.I. du Pont continued to head the firm until his sudden death, possibly from a heart attack, on October 31, 1834, at the age of 63. His son-in-law James Antoine Bidermann (1790-1865) then became director and senior partner, with E.I.'s sons Alfred Victor du Pont (1798-1856) and Alexis Irénée du Pont (1816-1857) as partner and superintendent, respectively. Bidermann, the son of one of the company's first stockholders Jacques Antoine Bidermann (1751-1817), had initially arrived from France in 1814 to investigate company affairs and to report on a dispute that had developed between du Pont and Peter Bauduy (1769-1833), another stockholder. Upon completing his investigation, Bidermann joined the company as E.I. du Pont's assistant and married Evelina du Pont (1796-1863) in 1816.

As the director, Bidermann reorganized the firm into a partnership split among E.I. du Pont's seven children and retired as senior partner in 1837, at which point Alfred Victor assumed the role. With the demand for powder continuing to grow as the United States expanded westward and as canal and railroad transportation networks were built, the company expanded further on the Brandywine, opening a Lower Yard downstream from the existing mills in May 1839. The Mexican-American War brought additional government orders, and in 1849, the company ventured to the West Coast to establish an agency in California to meet demand spurred by the California Gold Rush. Although demand and sales continued to increase, Alfred Victor du Pont struggled to maintain a firm hold on the company's finances, and an April 1847 explosion in the Upper Yard rocked the company further, killing eighteen people and causing the worst property loss to date.

Considering these difficulties, Alfred Victor du Pont retired as senior partner in January 1851 and was succeeded by his brother Henry du Pont (1812-1889), with Alexis I. du Pont and Eleuthère Irénée du Pont, II (1829-1877, eldest son of Alfred Victor) as junior partners. Having begun work for the company in 1834, Henry would remain as head of the company from 1851 until his death in 1889. After reviewing the company's books, Henry refocused the company's efforts on reducing production costs while increasing sales and was able to bring the company out of debt within a decade.

While Henry controlled financial matters for the company, he came to rely on the chemistry skills of his nephew Lammot du Pont (1831-1884), son of Alfred Victor, in matters of manufacturing. Lammot joined the company in 1849 after completing his studies in chemistry at the University of Pennsylvania. He introduced multiple improvements to the yards and was made a junior partner in the firm in 1857. Also, in 1857, Lammot patented B blasting powder, using Chilean sodium nitrate instead of the previously used potassium nitrate (saltpeter) from India. This process would later influence the company's decision to purchase lands in Chile. In 1859, the company purchased the powder mills of Parish, Silver & Co., on Big Wapwallopen Creek, Luzerne County, Pennsylvania. Lammot supervised the rebuilding of these mills to produce blasting powder, which was the first one owned by the company outside of Delaware.

In 1853, during the Crimean War, the DuPont Company secured its first contracts negotiated with foreign governments for gunpowder. During the American Civil War, the company became the largest supplier of powder for the United States military, thanks in part to Lammot du Pont's successful negotiations with Britain in 1862 to secure saltpeter. In 1872, in response to large fluctuations in the gunpowder industry after the war, the DuPont Company and several of its largest competitors organized the Gunpowder Trade Association, with Lammot du Pont as its president. In 1876, the DuPont Company secretly acquired one of its two largest competitors, the Hazard Powder Company, and throughout the 1870s and 1880s, the DuPont Company bought out hundreds of smaller competitors to consolidate its control of the market.

In 1868, Alfred Nobel (1833-1896) patented dynamite in the United States, and Lammot du Pont soon took a keen interest in dynamite research and production. In 1880, he organized the Repauno Chemical Company to manufacture dynamite at a new plant at Thompson's Point, New Jersey. Four years later, however, Lammot was killed in an explosion at the plant, but the company continued with the DuPont Company as its largest shareholder.

Upon Henry du Pont's death in 1889, the company's management passed to Eugene du Pont (1840-1902) and his brother Francis Gurney "Frank" du Pont (1850-1904). In 1890, at the request of the U.S. government, the DuPont Company started scientific research on smokeless gunpowder, also referred to as guncotton, and soon began the manufacture of it at its new works at Carney's Point, New Jersey. In 1897, inventor and chemist Hudson Maxim (1853-1927) sold many of his explosives and smokeless gunpowder patents to the DuPont Company, and Maxim continued research on explosives for the company through the early twentieth century. Although the company was entering other product markets, its primary product, black powder, was losing prominence, and the company's mills were becoming obsolete. In 1890, its original mills on the Brandywine suffered its largest explosion, which killed fourteen people and caused a million dollars worth of damage.

In 1899, the company dissolved its original partnership and incorporated it to redistribute the company's increasingly complex executive responsibilities. Eugene du Pont became president of this new firm but unexpectedly passed away in 1902. With no tenable options for a successor to the presidency, the remaining officers decided to sell the company. Three du Pont cousins, Alfred I. du Pont (1864-1935), T. Coleman du Pont (1863-1930), and Pierre S. du Pont (1870-1954), were able to purchase the company to keep management within the du Pont family. E. I. du Pont de Nemours & Co. was reincorporated on February 26, 1902.