Williams fought back after losing the opening set to take her singles match against Chanelle Scheepers 4-6 6-2 6-3, but the match was forced to a deciding mixed doubles clash as Isner lost his singles 7-6(0) 7-6(5) to Kevin Anderson.
Williams and Isner were a class apart in the doubles, however, breezing through to win 6-3 6-2.
The defeat means that the South African team, who also lost to Spain on the opening day, are out of the tournament.
The day's other match, between France and Spain, was also decided by the mixed doubles where Spain came through to win.
Jo-Wilfried Tsonga put France ahead as he beat Fernando Verdasco 7-5 6-3, but Spain's Anabel Medina Garrigues levelled the match with a 6-3 6-2 win over Mathilde Johansson.
The Spanish duo held firm, however, cruising to a 6-3 6-3 win in the doubles to claim the match 2-1.