Following on from his Atlanta title win, Isner made it back-to-back losses as he was beaten 6-4 6-7 (4-7) 6-3 by Croatian Ivan Dodig.
The big-serving American 10th seed sent down 13 aces, but he was unable to trouble Dodig in the deciding set on a rain-interrupted day in Toronto.
Dodig lost just two points on serve in the third set, breaking once to complete his victory in one hour 52 minutes at the ATP 1000 event played on hard courts.
French 13th seed Tsonga fared much better, seeing out a 7-6 (7-3) 6-1 win over countryman Edouard Roger-Vasselin.
Awaiting Tsonga in the second round is another Frenchman in Jeremy Chardy, who beat Argentine Federico Delbonis 6-3 5-7 6-4.
Croatian Cilic, the 15th seed, made it five wins in six meetings against Denis Istomin, beating the Uzbek 6-2 4-6 7-5.
He will have to wait to find out his next opponent as darkness saw the clash between Spain's Guillermo Garcia-Lopez and Malek Jaziri suspended with the Tunisian lucky loser leading 6-4 5-5.
Along with Isner, the other seed to fall on Monday was Roberto Bautista Agut after the 14th seed slumped to a 7-6 (9-7) 6-3 loss to fellow Spaniard Feliciano Lopez.
Lopez will face Tim Smyczek after the American eased past German Tobias Kamke 6-3 6-2 in a battle between two qualifiers.
Canadian wildcard Peter Polansky upset Pole Jerzy Janowicz 7-6 (7-5) 6-4, while Australian veteran Lleyton Hewitt committed 30 unforced errors in a 6-1 6-2 loss to France's Julien Benneteau.
South African Kevin Anderson overcame a slow start to get past Australian qualifier Thanasi Kokkinakis 4-6 6-2 6-3 and American Michael Russell was a 6-3 6-3 winner over Frenchman Nicolas Mahut.
French qualifier Benoit Paire battled past Colombian Alejandro Falla 6-7 (3-7) 6-4 6-2.