Conodoguinet Creek, a tributary of the Susquehanna River, crosses the center of the township.

Unincorporated communities in the township include Donnellytown in the northeast, Carlisle Springs in the northwest, and part of the Schlusser census-designated place along the western border.

The borough of Carlisle, the county seat, is approximately 4 miles (6 km) southwest of the highway crossing area via US 11, and Harrisburg, the state capital, is about 16 miles (26 km) to the east.

The Appalachian Trail crosses the northern half of the Cumberland Valley following a route near the eastern border of Middlesex Township.

20.0% of all households were made up of individuals, and 7.0% had someone living alone who was 65 years of age or older.

Out of the total population, 10.0% of those under the age of 18 and 4.6% of those 65 and older were living below the poverty line.