Born in Geneva, du Simitiere's original name was Pierre-Eugene Ducimetiere or Pierre-Eugene du Cimetiere. After leaving the Republic of Geneva, he spent more than a decade in the West Indies before moving to New York and then Philadelphia. He spelled his name Pierre-Eugene du Simitiere, Pierre Eugene du Simitiere, or du Symitiere after settling in Philadelphia. Elected to the American Philosophical Society in 1768, he further became one of its curators (1777-1781).
Du Simitiere served as the artistic consultant for the committees that designed the Great Seal of the United States, and in 1776 he submitted the first proposed design to include the Eye of Providence, which element was eventually adopted an suggested the adoption of the U.S. motto E pluribus unum ("Out of Many, One").
During the early Revolution, he drew portraits of many American military and political leaders, including Friedrich Wilhelm von Steuben, John Jay, William Henry Drayton, John Dickinson, Benedict Arnold, among others, all of which were published in a 1783 book.