Mattek-Sands upset French Open champion Francesca Schiavone 6-4, 6-4 to put the US ahead and Isner then beat Potito Starace 7-6, 4-6, 6-4 to make it 2-0 before the Italians won the mixed doubles 6-7, 6-2 10-3.
The result leaves the United States in pole position to qualify for the final from Group B, though all four teams can still make it. Great Britain take on France later on Wednesday.
World number seven Schiavone looked out of sorts against Mattek-Sands, who wore a snazzy all-black outfit, topped off with long black socks and a rose tattoo on the underside of her right arm.
The American used her speed to great effect and showed some useful skills at the net as she denied Schiavone the opportunities to attack she enjoys so much.
One break was enough to win the first set and she then recovered from a break down to win the second for a surprise victory over a player ranked 51 places above her.
Isner edged out Starace in a tough three-set battle. The American looked tired towards the end of the second set and Starace took advantage to level the match and then forge a 3-1 lead in the decider.
But Isner found his energy levels just in time to win four straight games and then serve out for victory, finishing with his 23rd ace.
The Americans looked on course for a clean sweep when they won the first set of the mixed doubles on the tiebreak but Starace and Schiavone hit back well to claim what could yet be a vital point.