Thomas Knowlton joined his militia, the Ashford Company, which became part of the 5th Connecticut Regiment, along with the men from Windham, Mansfield and Coventry, Connecticut. Knowlton was chosen unanimously as captain and led 200 men into Massachusetts. His force consisted of farmers, without uniforms, primarily armed with shotguns.

Knowlton was ordered to Charlestown to join Colonel William Prescott. Knowlton's troops were sent by Colonel Prescott to oppose the advancing British grenadiers, and took their posts on the side of Breed's hill. Knowlton was in command of a 200 men work party. His men dismantled another fence further back and used its rails to strengthen the existing fence, and they filled the space between with new-mown grass which formed an effective breastwork. There they held their ground until the general retreat, and were among those providing cover as the troops retreated. Only three men from Knowlton's company died in the battle.

In 1776, Knowlton was sent by General Washington during the Siege of Boston to burn any remaining buildings at the base of Bunker Hill, with orders to capture any British troops guarding the location. Knowlton accomplished his mission without firing a shot or losing a single man