St. Mary's roots go back to 1741 when a small mission chapel was established in "Barnstown" under the guidance of the Jesuit missionaries who served St. John's Parish in Frederick, Maryland.

After fleeing France during the French Revolution, Father John Dubois became the pastor of St. John's Parish. In 1807, Father Dubois recognized the growth of the mission and proposed to Bishop John Carroll of Baltimore that a Catholic Church be built in upper Montgomery County. "Barnstown" was renamed "Barnesville" and became the site for the new parish. The church immediately enjoyed community involvement with Zachariah Knott donating 8 acres of land and another local parishioner donating nearby timber for its construction. Fr. Dubois moved on to Emmitsburg where he established Mount St. Mary's and was also spiritual director to St. Elizabeth Ann Seton. Later, as bishop of New York City, he ordained St. John Neumann to the priesthood. Because these two American saints were directly influenced by Fr. Dubois, they are depicted with him over the entrance to the present church.

Behind the church, St. Mary's cemetery is the site of mass burials of those C & O Railroad builders who succumbed to the Cholera epidemic. The cemetery also became the final resting place of many Civil War soldiers. Barnesville saw action during the Civil War as it changed hands between Confederate and Union soldiers at least three times.

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