No. 8-seeded Jack Sock, the highest ranked of the American men, also lost 6-1, 7-6 (4), 5-7), 6-3 to Yuichi Sugita.
Ebden broke Isner's serve in the final game, clinching the match when Isner netted an attempted backhand half-volley.
In other first-round action, third-seeded Grigor Dimitrov advanced to the second round for the seventh time with a 6-3, 6-2, 6-1 win over qualifier Dennis Novak.
Last year at Melbourne Park, Dimitrov equaled his best Grand Slam result, reaching the semifinals before losing to Rafael Nadal in five sets. He also lost a Wimbledon semifinal in 2014 to Novak Djokovic.
Dimitrov has never lost to a qualifier in seven matches at a Grand Slam tournament, and he has dropped only one set.
Earlier, US Open finalist Kevin Anderson?lost in five sets to Kyle Edmund on Show Court 3, becoming the first seeded man to lose at the Australian Open.
The 11th-seeded Anderson twice led by a set but couldn't fend off Edmund, who got the decisive break in the seventh game of the fifth set and served out at love for a 6-7 (4), 6-3, 3-6, 6-3, 6-4 win.
It was the only meeting since Anderson beat Edmund in five sets in the third round of the French Open.
Ryan Harrison was among the other men advancing, beating Dudi Sela in five sets, along with No. 6 Marin Cilic, No. 10 Pablo Carreno Busta, No. 23 Gilles Muller, No. 24 Diego Schwartzman, No. 28 Damir Dzumhur, No. 30? Andrey Rublev?and No. 31 Pablo Cuevas.
No. 27 Philipp Kohlschreiber lost to Yoshihito Nishioka in five sets.
Information from The Associated Press was used in this report.