David Ferrer put the hosts ahead in the best-of-five series with a 4-6, 6-2, 6-2, 6-4 win over Querrey. Nicolas Almagro beat Isner 6-4, 4-6, 6-4, 3-6, 7-5 to leave Spain one point from its fourth final in five years.
Almagro overcame 25 aces from Isner, who saved three match points before hitting a forehand long.
The U.S. must win the doubles match Saturday to stay in the series, with brothers Mike and Bob Bryan facing Marcel Granollers and Marc Lopez. The Bryan twins are 4-0 when the U.S. has faced a 2-0 deficit, and 5-0 against Spain.
The United States has only rallied back to win from a 0-2 deficit once in 38 series -- in 1934 against Australia. Since the start of World Group play, Spain is 37-0 after winning both opening singles.
The winner plays either Argentina or the Czech Republic in the final in November.
Almagro and Ferrer are a combined 23-1 on clay. While Querrey wasted nine break opportunities in the fourth set of his match, Isner took advantage of his lone chance in the fourth to reach the deciding set.
As the light quickly faded, neither player relented on serve. Isner fired another ace to save a 16th break point before committing three unforced errors in the final game.
Ferrer started well with a break but eventually surrendered his only two service games of the match to lose the first set to Querrey.
Querrey, returning to Davis Cup play for the first time since 2010, failed to convert 12 break chances as the wind gusted off the Bay of Biscay.
"On a couple of them, I should have been more aggressive but he played good points," said the 26th-ranked Querrey. "I feel if I get one of those breaks, it could be a different game."
Ferrer played on hard courts in the U.S. Open semifinal on Sunday.
"It's never easy to adapt from hard court to clay, and with only three to four days even less," said Ferrer, who is 15-0 on clay in the competition. "I didn't feel too bad, but I didn't play perfect tennis, either."