Isner evened the title contest 1-1 with a 6-3, 6-4 win over Ruben Bemelmans after Henin won the final five games of her match to beat Mattek-Sands 7-6 (6), 6-3.
Belgium advanced as a surprise finalist from Group A when first-place finisher Serbia could not play the final due to an abdominal injury to Ana Ivanovic.
Isner's booming serve and all-court cover put the Americans in control in the mixed doubles decider. Mattek-Sands replaced Serena Williams, who pulled out of the American team in late November due to her recovery from a foot injury.
"It's been an amazing week, my first visit to Perth, and I couldn't have finished it in a better way," Mattek-Sands, who was attired in her usual brightly colored dress, said at the trophy presentation.
Isner said after his singles win that he didn't know what to expect against Bemelmans.
"I didn't really know too much about his game," Isner said. "My game plan was to go out there and try to make a lot of returns and make him play.
"He's kind of a flashy player so he's going to go for his shots, but I also know he's going to make a few mistakes here and there."
Henin, who found out late Friday she would be playing in the final, said she struggled mentally to prepare for the match.
"I was in my room and ... they told me 'you will play the final tomorrow'," Henin said.
"It was strange because mentally I was really focused on leaving for Melbourne on Sunday. I rested yesterday for my elbow and today I started a bit slowly but I thought Bethanie put a lot of pressure on my serve and I wasn't aggressive enough on my serve.
"Then in the second ... the last five games I was feeling more aggressive."
Henin missed the second half of last year with an elbow injury and said she was still feeling some pain in the joint.
"I have to deal with it," she said. "It's OK, I can play, but sometimes my focus is a lot on it. I'm going to work on it more and I'm glad with the four matches I've had (in Perth)."