The area around Middlesex Fells is known to have been explored by John Winthrop, Governor of the Massachusetts Bay Colony, in 1632. The reservation's lands have been used for the production of timber, granite, and ice. Abundant water power meant that many mills, including one that manufactured some of the first vulcanized rubber products, were located here. Remnants of early mill works are visible in the Spot Pond Archeological District, located in the Virginia Woods section, the site of the former mill village of Haywardville.
The reservation was initiated in 1891 with the donation of Virginia Wood to The Trustees of Reservations by Fannie Tudor as a memorial to her daughter, Virginia. As a child Virginia loved to walk in the woods surrounding the Italianate mansion her grandfather had given her mother on Spot Pond in 1862. Virginia died in 1888 in Paris, where she had moved with her mother after the death of her father Henry in 1864; Fannie died in Paris a year after her gift. (The property was later donated to the Metropolitan District Commission in 1923.) In 1893, the state took the property over and began managing it as a state park.
At some point in the 1800s or earlier, a stone tablet was erected on Great Island, which reads "WHERE SHUTE FELL". There are several contradictory stories explaining the significance of this phrase.
Boston Regional Medical Center was located within the Reservation along Woodland Road in Stoneham, until it closed in February 1999.
In addition to being a state park, portions of the park and structures within it are listed on the National Register of Historic Places. The entire area surrounding Spot Pond to the east of I-93 is within the Middlesex Fells Reservoirs Historic District, and the roadways in the park and on its borders are listed as the Middlesex Fells Reservation Parkways. The park's visitor center on Woodland Road in Stoneham is in the historic John Botume House, which is not far from the 1906 Metropolitan District Commission Pumping House. Historically important archaeological sites in the park are listed as part of the Spot Pond Archeological District. Roadways connecting the park to other elements of the Metropolitan Park System are also listed; these include the Fells Connector Parkways, which connect the park to the Mystic River Reservation in Winchester, and the Lynn Fells Parkway, connecting the park to the Breakheart Reservation in Saugus.