In the years immediately preceding the war and during its first term of service, the regiment consisted primarily of companies from Essex County as well as Boston and Charlestown.

Their second term of service lasted 9 months from September 1862 to July 1863 during which they were stationed in New Bern, North Carolina, participated in several expeditions and saw minor combat including the Battle of Goldsborough Bridge.

Their third enlistment in response to the emergency call for troops to defend Washington, D.C. lasted 100 days from July to November 1864 during which they were stationed in various fortifications around Baltimore, Maryland, primarily in Fort McHenry.

[9] On July 21, 1861, just days before the end of their 90-day term of service, the 5th Massachusetts took part in the First Battle of Bull Run, the first major engagement of Civil War.

Before their departure from Alexandria, Major General Irvin McDowell, commanding the Union Army of Northeastern Virginia, addressed the 5th Massachusetts and, in light of their term of service being nearly at an end, offered them the option of foregoing the campaign.

[10] Assigned to the First Brigade (Franklin's), Third Division (Heintzelman's) of the Army of Northeastern Virginia, the 5th Massachusetts was among those units sent to probe the Confederate right flank on July 18 resulting in the Battle of Blackburn's Ford.

On July 21, precisely three months after they departed Boston, the 5th Massachusetts crossed Sudley Springs Ford and participated in pitched combat on Henry House Hill.

[11] The regiment advanced more than a mile from Sudley Springs Ford to Henry House Hill at the double-quick in full gear.

[7] When Confederate General Thomas "Stonewall" Jackson led a counterattack, the 5th Massachusetts retreated in disorder from Henry House Hill with the rest of the Union Army.

The objective of this maneuver was to disrupt the Confederate supply line along the Wilmington and Weldon Railroad by destroying the Goldsborough Bridge.

Over the course of this expedition, the unit marched 180 miles and took part in the Battles of Kinston, White Hall and Goldsborough Bridge.

[20] During the spring of 1863, the unit took part in several expeditions to reconnoiter and dislodge enemy positions along the Pamlico and Neuse Rivers.

[22] In the spring of 1864, as Major General Ulysses Grant prepared to launch his Overland Campaign, he removed fresh troops from the defensive fortifications of Washington and transferred them into the field to strengthen the Army of the Potomac.

The attack failed, however the fact that Confederate troops advanced to the outskirts of Washington D.C. caused widespread panic.

This prompted Lincoln to issue a call for 500,000 troops to serve a brief term of 100 days to bolster defenses around the capital.

[27] The regimental historian noted that their duties in Baltimore were focused more on maintaining order in a city known for its secessionist views rather than external threats.

[26] Soldiers of the 5th Massachusetts who died during their second term of service with the Department of North Carolina are buried in the New Bern National Cemetery.