In February 1754, Dinwiddie promoted Washington to lieutenant colonel and second-in-command of the 300-strong Virginia Regiment, with orders to confront French forces at the Forks of the Ohio. Washington set out for the Forks with half the regiment in April and soon learned a French force of 1,000 had begun construction of Fort Duquesne there. In May, having set up a defensive position at Great Meadows, he learned that the French had made camp seven miles (11 km) away; he decided to take the offensive.

The French detachment proved to be only about 50 men, so Washington advanced on May 28 with a small force of Virginians and Indian allies to ambush them. What took place, known as the Battle of Jumonville Glen or the "Jumonville affair", was disputed, and French forces were killed outright with muskets and hatchets. French commander Joseph Coulon de Jumonville, who carried a diplomatic message for the British to evacuate, was killed. French forces found Jumonville and some of his men dead and scalped and assumed Washington was responsible. Washington blamed his translator for not communicating the French intentions. Dinwiddie congratulated Washington for his victory over the French. This incident ignited the French and Indian War, which later became part of the larger Seven Years' War.

The full Virginia Regiment joined Washington at Fort Necessity the following month with news that he had been promoted to command of the regiment and colonel upon the regimental commander's death. The regiment was reinforced by an independent company of a hundred South Carolinians led by Captain James Mackay, whose royal commission outranked that of Washington, and a conflict of command ensued. On July 3, a French force attacked with 900 men, and the ensuing battle ended in Washington's surrender. In the aftermath, Colonel James Innes took command of intercolonial forces, the Virginia Regiment was divided, and Washington was offered a captaincy which he refused, with the resignation of his commission.