Settlement of the area began around 1640 with the county being officially formed in 1669 from a part of Lancaster County. The county's only incorporated town, Urbanna, was established in 1680 serving initially as a port for shipping agricultural products and later as the county's commercial and governmental center.
Rosegill Estate, a Middlesex County plantation first constructed in 1649, served as the temporary seat of the colony under two royal Governors of Virginia, (Sir Henry Chicheley, who served under Thomas Culpeper, 2nd Baron Culpeper of Thoresway, and Lord Francis Howard, 5th Baron Howard of Effingham). The home remains a private residence to this day, though the estate has been purchased by a Northern Virginia Development firm, and much of it is slated to become a 700 home subdivision. An archaeological survey of the ground underneath the planned development's first phase has revealed what appear to be parts of the Nimcock Native American village, and also parts of the Rosegill slave community. The developer intends to proceed with building houses over a portion of the artifacts, which will render excavation and study of them impossible.
During the American Civil War, Urbanna was initially planned as the point of landing for General George B. McClellan's 1862 Peninsula Campaign of 1862 to take Richmond, but ultimately, the failed campaign utilized Fort Monroe as its starting point, almost doubling the distance by land to the Confederate citadel. Delays in reaching the gates of Richmond allowed the Confederates ample time to erect substantial defensive batteries, contributing to the Union failure.
The Historic Middlesex County Courthouse was built in 1850-1874 by architects William R. Jones and John P. Hill, and is listed in the National Register of Historic Places. Construction of a new courthouse began in 2003 and was completed in 2004. It was not occupied until September 2007, however, due to a legal dispute between the county and the architect. The Historic Courthouse has been remodeled and now serves as the Board of Supervisors meeting room and the Registrar's Office.
Urbanna was incorporated on April 2, 1902, comprising an area of . The Town of Urbanna remains the county's largest commercial center and its only incorporated area but the county seat of government has moved to the Village of Saluda on U.S. Route 17. To the east, almost to Stingray Point, the Village of Deltaville is situated on State Route 33 between the mouths of the Rappahannock and Piankatank Rivers. Once a major center for wooden boat building, the village remains a commercial and recreational center. The waterfront east to Stingray Point is home to many marinas, with a heavy concentration on Broad Creek.