The AFL Commission will now rule on Jobe Watson's Brownlow Medal after he and 33 other Essendon players failed to have their doping bans overturned.
The Swiss Federal Tribunal has ruled against the current and past Bombers players.
Their appeal to the tribunal was the last legal step available to them.
They were challenging the Court of Arbitration for Sport ruling in January that went against the players.
It meant 17 players, including 12 at Essendon, were banned from playing this season.
With the appeal over, the AFL commission can now go ahead with its landmark hearing that decides whether Watson keeps his 2012 Brownlow Medal.
The supplements regime at Essendon that led to the bans operated during the 2012 season.
The AFL announced the hearing after the CAS verdict, but postponed it until the Swiss appeal verdict.
The likelihood is that the commission will take the unprecedented step of stripping Watson of his Brownlow.
Richmond captain Trent Cotchin and Hawthorn veteran Sam Mitchell finished joint runners-up behind Watson in the 2012 Brownlow voting.
Watson was the last of the banned players to confirm he would return to playing and he is now back at Essendon.
The banned players still at AFL clubs have now returned to training, with some restrictions.
When he made his announcement on September 23, Watson said he was not thinking about the prospect of losing the Brownlow.
"If you spend your life living in that world of speculation, you miss the moment, and I've removed myself from it and I've just lived in moments, and that was liberating for me," he said.
"(The Brownlow) doesn't define me."
The AFL Players Association, Essendon and the AFL were yet to comment publicly on Tuesday night about the Swiss appeal verdict.
The verdict was handed down in German.
Watson has been invited to speak to the commission but it wasn't immediately clear when the hearing would be held.
In May, AFL chief executive Gillon McLachlan admitted there was a collective feeling of dread about having to make the call on Watson's Brownlow.
"I think the people charged with the responsibility of making that decision will not have made a more difficult decision - not just in their time in football, but almost in their lives," he said.
"I don't want to over-dramatise it, but that will be as hard a decision as anyone on the commission has had to make, I'm sure of it."
Apart from Watson's Brownlow, another issue yet to be resolved from the long-running supplements saga is the banned players' negotiations with Essendon over financial compensation.
The Essendon supplements saga is the biggest scandal in AFL history.
Bombers coach James Hird was suspended for a season because of it.
He returned to the job, but was sacked late last season.
The AFL banned Essendon from playing in the 2013 finals at the same time it suspended Hird.
The doping bans meant the Bombers' makeshift team finished last this season.
An AFL anti-doping tribunal initially cleared the 34 players after an ASADA investigation.
But WADA took the case to CAS and won, leading to the Swiss appeal.
