Rev. Gloucester founded his congregation in the Hall Building, on the intersection of Fulton and Cranberry Streets. Gloucester's father, who was a Presbyterian minister in Philadelphia, inspired him to found his own religious society in Brooklyn in 1849.

The church moved to 106 Prince Street between Myrtle and Willoughby Avenues sometime after its founding. It was at this location that the church was a place of refuge for those fleeing slavery. In later years, the church became a meeting place for the Literary Union, a group working to improve the civil rights of African Americans in the public education system, advocating for desegregation and the hiring of black educators. Though some of the Union's members were followers of the church, many were not. Siloam's welcoming of all community groups working to implement change is part of the congregation's legacy.

By the early 1900s, the Prince Street location of Siloam Presbyterian had fallen into disrepair, and the building was demolished. While raising funds to rebuild, the congregation continued to meet in various spaces. Once the congregation secured enough funds to rebuild and expand their church, the city announced plans for a Manhattan Bridge Approach that would encroach on their site. The church continued to meet, but it did not settle on a long-term location for a number of years.

One of the locations that the Siloam Presbyterian congregation met in was the A.M.E. Zion Memorial Church. A.M.E. Zion, another historically Black church, shared it space with the other congregation in 1907.

In 1910, Siloam moved to 404-8 Lafayette Avenue, formerly First Church of Christ, Scientist.

After years of meeting in various congregation halls and community spaces, Siloam Presbyterian Church moved to the former Central Church on Jefferson Avenue in 1944.

The building had been recently rebuilt, as the 1909 Central Church, constructed in 1909, was destroyed by a fire in 1936.

At 260 Jefferson Street, Siloam Presbyterian continued its legacy as a site that pioneered activism and social reform. Reverend Milton Galamison, who led the church, served as a member of the Brooklyn Branch of the NAACP. He founded the Parents' Workshop for Equality in New York Schools, which strove to end racial discrimination and offer all children equal opportunities namely Black and Puerto Rican students who had been disenfranchised by the New York Public School System.

In 1959, Siloam hosted the "Moslem Dinner and Surprise Program," a program led by Malcolm X who, at the time, was a member of the Nation of Islam. Galamison, of Siloam, was the leader of one of the largest Presbyterian congregations in the predominantly African-American neighborhood of Bed-Stuy. By opening his doors to another religious group, Rev. Galamison "situated the Muslims...in a place encoded by a tradition that shared the NOI’s commitment to anti-racist work," according to Zaheer Ali in the African American Intellectual Society's "Black Perspectives." This move united the two leaders and was exemplary of how "local conditions placed different activists and their communities into shared spaces that offered the potential for the exchange and critique of ideas." In 2019, Siloam Presbyterian Church Celebrated its 170th year anniversary.