The American, who prevailed over 11 hours split across three days when he beat Nicolas Mahut in 2010, found himself going past the four-hour mark against his French opponent.
Isner led by two sets to one when play was suspended on Saturday evening and could not finish the job when proceedings resumed on middle Sunday.
His opponent dominated the fourth set before the fifth saw neither man able to make any impression with their returns over the opening 30 games.
Isner's chance came at 16-15 but Tsonga saw off a match point in confident style before making the decisive breakthrough himself two games later.
The final scorecard read 6-7(3) 3-6 7-6(5) 6-2 19-17 after four hours and 24 minutes of action on No.2 Court.