Tsonga, whose Masters tournament win in Toronto on Sunday was his second career success at ATP 1000 level, was humbled by Russia's Mikhail Youzhny 6-1 6-4 in the first round in Cincinnati.
The Frenchman edged back into the ATP's top 10 after beating Roger Federer in the Toronto final, but Tsonga's booming serve let him down against Youzhny.
Tsonga served at 52 per cent, but he won just seven of 23 points behind his second ball as the 25th-ranked Youzhny progressed.
Youzhny now has a positive head-to-head record against Tsonga, although the win was only his second via match point - accounting for retirements.
Tsonga was the only seeded casualty in Ohio, as top seed Djokovic and third seed Stan Wawrinka both negotiated their second-round matches.
Serbia's Djokovic staved off the plucky Gilles Simon of France 6-3 4-6 6-4, while Swiss Wawrinka dispatched of German qualifier Benjamin Becker 6-3 7-6 (7-5).
The pair are the first players through to the last 16 of the hard-court tournament, which serves as the penultimate warm-up event to the US Open starting later in August.
In more first-round results, seeds Ernests Gulbis (nine), Roberto Bautista Agut (13) and Marin Cilic (14) advanced.
Latvia's Gulbis took two hours to down Croatian Ivan Dodig in straight sets, winning 7-6 (10-8) 7-6 (7-4).
Bautista Agut, of Spain, clawed his way past Slovakian lefty Martin Klizan 6-2 4-6 7-5, while Croatia's Cilic dismissed another left-hander, Spaniard Feliciano Lopez, 6-3 6-4.
Frenchman Julien Benneteau saved two match points to fend off lucky loser Blaz Rola of Slovenia 6-7 (7-9) 6-3 7-6 (9-7).
Similarly, Canada's Vasek Pospisil denied Czech veteran Radek Stepanek thrice on match point before posting a 5-7 6-1 7-6 (7-4) triumph.
American wildcards Robby Ginepri and Steve Johnson both capitalised on their chances in the main draw with first-up wins.
Ginepri battled past rising Austrian star Dominic Thiem 6-3 5-7 7-5, while Johnson was handed a straightforward 6-4 6-1 win over Benoit Paire courtesy of a poor performance from the French qualifier.
Italy's Andreas Seppi overcame a deficit to defeat Colombian Santiago Giraldo 4-6 6-2 7-5.
Down a set and at 2-2 in the second, Seppi reeled off eight straight games to earn a 4-0 lead in the decider - only to narrowly avoid a tie-break as Giraldo finished strongly.
British qualifier James Ward hit eight aces as he defeated Spain's Guillermo Garcia-Lopez 6-2 3-6 6-1.