The Catalans were punished back in April after they were found to be in breach of art. 19 of Fifa regulations - which states international transfers of players are only permitted if the player is over the age of 18, bar three exceptions - in the case of 10 minor players, while they were also found guilty of further infringements.
Fifa released a statement on Wednesday to confirm they had rejected Barca's appeal, meaning they would now be forced to see out two consecutive transfer windows, beginning in January 2015, without being able to complete any signings.
"The Fifa Appeal Committee has decided to reject the appeals lodged by Barcelona and the Real Federacion Espanola de Futbol (RFEF) and to confirm in their entirety the decisions rendered by the Fifa Disciplinary Committee in the respective cases relating to the protection of minors," a Fifa statement reads.
"As such, Barcelona are to serve a transfer ban which will see the club prevented from registering any players at both national and international level for two complete and consecutive transfer periods, starting with the next registration period (January 2015) given that the appeal of the club had been granted suspensive effect by the chairman of the Fifa Appeal Committee.
"Barcelona have also been ordered to pay a fine of CHF 450,000 (€371,000) and been given a period of 90 days from August 20 in which to regularise the situation of all minor players concerned."
Barca, however, have insisted they will continue to fight the sanction by referring the case to CAS.
"Following the resolution announced today by the Fifa Appeals Committee on violations of regulations regarding the transfer and registration of children aged under 18 years, FC Barcelona announces that it shall continue defending its interests before the highest sporting authority, in this case the Court of Arbitration for Sport (CAS)," a statement reads.
"FC Barcelona may not in any way share a resolution that is an affront to the spirit of our Masia, a world renowned example of academic, human and sporting education."
The Camp Nou side's appeal against the ban was initially granted suspensive effect, allowing them to secure the signings of Luis Suarez, Ivan Rakitic, Jeremy Mathieu, Thomas Vermaelen, Marc-Andre ter Stegen, Claudio Bravo and Alen Halilovic this summer.
Meanwhile, Fifa's Appeal Committee has also upheld the Spanish Football Federation (RFEF)'s punishment in the same case.
With regard to the RFEF, the Disciplinary Committee previously determined that the association had also violated art. 19 of the regulations and other provisions in the context of the transfer and first registration of certain minor players.
"The RFEF has been ordered to pay a fine of CHF 500,000 (€413,000) and granted a period of one year in which to regularise their regulatory framework and existing system concerning the international transfer of minors in football," the Fifa statement continues.
"The terms of the decisions taken by the Fifa Appeal Committee were communicated to Barcelona and the RFEF on Wednesday."