At the outbreak of the Civil War, the six foot tall Penrose mustered in with what was to become the Sixth Reserves, 35th Regiment Pennsylvania Volunteers and was elected as a lieutenant-colonel when the unit was organized at Harrisburg in June 1861.  The 35th was accepted into federal service on July 27, five days after moving to Camp Tenallytown; it wintered at Camp Pierpoint near Langley, Virginia.  Penrose, as temporary commander of the regiment, saw action and was commended after the battle of Dranesville on December 20, 1861 for his coolness in command and the 35th Regiment's pursuit of the enemy.  Camp Pierpoint, unfortunately, was a poorly drained establishment that brought widespread sickness, mostly probably malaria, to the regiment, including Penrose.  With the regrets of his brigade commander, General Ord, Penrose resigned his commission due to illness early in 1862 and returned to Carlisle.

Penrose sat on the town council in 1862 and 1863 and it was he and several of his fellow members that went to the southern edge of town in June 1863 to discuss with the second wave of Confederate troops to invade Carlisle a peaceful treatment for the town.  Confusion led to the brief shelling before the occupation, however. Following the war, the respected and admired Penrose continued his popular law practice. 

In 1858, Penrose married Valeria Merchant of Pittsburgh, the daughter of an army general.  The couple had four daughters, Sarah, Valeria, Ellen, and Jennie.  The illness contracted in the Union Army camps never left him, however, and William McFunn Penrose died in Carlisle at his home on High Street after a short illness on September 2, 1872.  He was forty-seven years old.