Stephen Mattoon was born in Champion, New York on May 5, 1815 or 1816 (sources agree on the date but not the year of his birth). He graduated from Union College in 1842 and went on to the seminary at Princeton. During his years there he met Mary Lourie, preceptress of the Argyle Academy. In February of 1846 Mattoon was ordained by Troy Presbytery; in June he and Mary Lourie were married, and in July they sailed for Siam under the auspices of the Board of Foreign Missions (PCUSA). They arrived in Bangkok in March of 1847.

The Siamese language fascinated Mattoon, and his missionary work involved translation and distribution of Christian literature. His chief project was a complete Siamese New Testament, which was not finished until he left Siam in 1866. He served as pastor of the First Church in Bangkok from 1860 to 1866. Mattoon was greatly esteemed by the Siamese court; in recognition of this, the U.S. Minister Plenipotentiary appointed him the first United States Consul in Bangkok. He held this position from 1856 until 1859. Mary Lourie Mattoon taught English and took Siamese girls and young women into her home, instructing and training them as Christians. She also gave birth to three children during these years: Lourie (1850-1851), Mary Lourie (b. 1854) and Emma Williams (b. 1857). The Mattoons also adopted a Siamese child, whom they christened Esther.

Mary Lourie Mattoon's frail health forced her to leave Siam in 1864. Stephen Mattoon remained in Bangkok to finish his translation of the New Testament. In 1866 he rejoined his family in New York, accepting assignment as pastor of the church at Ballston Spa. In 1870 he was asked by the Freedmen's Board to take over the presidency of Biddle Institute (now Johnson C. Smith Seminary) in North Carolina. He served Biddle as its president until 1885 and as professor of theology until his death in 1889.