The US team will now await their final opponents after completing a clean sweep of victories at Perth Arena on Thursday, which started with Williams coming through a three-set contest with Lucie Safarova.
Despite saving two set-points in the penultimate game of the first set, Safarova was behind after 38 minutes but levelled after a hard-fought tie-break in the second.
However, Williams - seeking to become the first player to win three Hopman Cup titles - showed her steel in the final set to clinch a 6-3 6-7 (1-7) 7-6 (8-6) win and get the US up and running.
"I never give up," she explained. I've been through so much in my life and I just keep fighting."
Isner then saw off Adam Pavlasek 7-6 (7-4) 6-4 to keep American momentum going before a crucial deciding match-up in the mixed doubles.
Victory for the Czechs would have seen them qualify at their opponents' expense.
However, Isner and Williams eased to their second victories of the day to book a place in the final.
Asked whether the pair were the best mixed team ever, Isner responded: "I don't know about that. We're pretty good. Serena is arguably the best player of all time, so that doesn't hurt."
The day got under way with Canada's Eugenie Bouchard taking on Italian Flavia Pennetta in their first career meeting - the former winning through 6-3 6-4.
In the men's singles, Vasek Pospisil completed Canada's victory in the tie courtesy of a comprehensive 6-0 6-3 win over Fabio Fognini.
However, Italy denied their opponents a clean sweep and any hope of a final place, as Fognini and Pennetta claimed the mixed doubles with a 6-3 4-6 10-7 success.
Great Britain, France and Poland will compete to fill the other final spot on Friday.