The architecture of the church, which opened on March 12, 1862, is noteworthy. It is one of the finest examples of early Romanesque Revival church architecture in New York (the mode had been inaugurated in the 1840s by the architect Richard Upjohn at the Congregation Church of the Pilgrims on Remsen and Henry streets in Brooklyn Heights.) The simplicity and, some would say, primitivism of the Romanesque style commended itself to dissenting Protestant sects that objected to the Gothic style embraced by high Episcopalian and Roman Catholic congregations for whom sacramental ritual rather than preaching was the main point of the worship service. Lafayette Avenue Presbyterian Chruch adopted a semi-circular, "theatrical" seating plan, influenced by that of Henry Ward Beecher's Plymouth Church (1849), which was felt better to serve congregants of a gifted preacher.

In 1976, under the Reverend George Litch Knight, who was Lafayette's pastor for 33 years, the church interior was significantly altered by the incorporation of murals, "Mighty Cloud of Witnesses," painted by Hank Prussing, who was then 27 years old and a graduate of nearby Pratt Institute. For the Lafayette Avenue commission, he took 500 photographs of neighborhood scenes and residents as preparation for a mural sequence that depicts vividly observed vignettes of neighborhood life and characters. Its a startling thing to see when you enter the church, but the murals proved popular with many congregants, who even recognized local faces among Prussing's subjects.