The congregation began services and formally organized in 1844. The first building was occupied in 1851, with subsequent building programs in 1915, 1936, 1949, 1977 and 1988. John W. Brubacher is considered the founding leader of the group. The congregation originated through immigration from Pennsylvania.
The meetinghouse was located west of St. Jacobs just north of the intersection of Regional Road 17 and Township Road 32 at the present location of Three Bridges School and the congregation's cemetery. At that time it was known as the Conestoga Mennonite Meetinghouse. From 1889 to 1892 the congregation shared a building with the Old Order Mennonites who retain the Conestoga Mennonite Meetinghouse name. In 1915 the congregation moved into the town of St. Jacobs and changed its name to St. Jacobs Mennonite Church. The congregation transitioned from German to English in the 1910s.
In 1925 there were 149 members; in 1950, 364; in 1965, 412; in 1975, 403; in 1985, 425; in 1995, 346; in 2000, 346; in 2010, 374. The congregation has been affiliated with the Mennonite Conference of Ontario (1844-1988), Mennonite Church (1898-), Mennonite Church Eastern Canada (1988-) and Mennonite Church Canada (1995-).