He was the eldest son of Karl William, Prince of Anhalt-Zerbst, by his wife Sophie, daughter of August, Duke of Saxe-Weissenfels.

In 1718, after the death of Karl William, John Augustus became prince of Anhalt-Zerbst.

John Augustus married Fredericka (b. Gotha, 24 March 1675; d. Karlsbad, 28 May 1709), daughter of Frederick I, Duke of Saxe-Gotha-Altenburg, on 25 May 1702 in Zerbst.

He married a second time to Hedwig Fredericka (b. Weiltingen, 18 October 1691; d. Zerbst, 14 August 1752), daughter of Frederick Ferdinand, Duke of Württemberg-Weiltingen (a grandson of Julius Frederick, Duke of Württemberg-Weiltingen), on 8 October 1715 in Zerbst.

Since John Augustus died without issue, the elder line of Anhalt-Zerbst became extinct.