The Swiss Federal Court dismissed the Essendon 34's appeal on Tuesday evening and upheld the Court of Arbitration for Sport's ruling to find the players guilty of doping offences relating to their 2012 supplements regime.
The 34 suspended Bombers' were initially cleared by the AFL anti-doping tribunal in March 2015, however, were then found guilty by the Court of Arbitration for Sport in January 2016 and suspended for two years, with 12 months of the ban backdated.
The players, while resigned to the fact they would not play this season, appealed to the Swiss Federal Court in a bid to clear their names.
With the appeal failing, 2012 Brownlow Medalist, Jobe Watson, looks all but certain to be stripped of the award, with joint runners-up Trent Cotchin and Sam Mitchell likely to be awarded the medal. However, the AFL Commission is yet to make a formal ruling on the matter.
Essendon chairman Lindsay Tanner released the following statement on Tuesday night.
"The club respected and supported the players' decision to exercise the only legal right to appeal they had in this process," Tanner said.
"We maintain our view that the decision and penalty handed down by the Court of Arbitration for Sport was manifestly unfair on our players.
"The club will not be making any further comment at this point in time."
The AFL Players Association also released a brief statement acknowledging the appeal finding.
"The decision is written in German and it must now be translated into English to allow the lawyers for the players to review the reasons of the Court," the AFLPA said.
"As such we are not in a position to elaborate any further on the Court's decision at this point in time."