Barcelona were unsuccessful in their appeal of Neymar's three-match ban before the appeals committee of the Royal Spanish Football Federation (RFEF) on Thursday.
The Blaugrana club have one more option and will take the case before the federation's Court of Arbitration for Sport on Friday. If they win, Neymar could be eligible to play in Sunday's Clasico against Real Madrid.
Neymar was sent off for picking up two yellow cards as Barca were beaten at Malaga earlier this month, becoming their first player to be dismissed in La Liga since October 2015. He was later dealt the three-match ban
The dismissal was Neymar's first in his four seasons with the club.
His first booking was for obstructing the taking of a free-kick by tying his laces and the second was for a late challenge on Roberto Rosales.
That carries a one-match ban -- but referee Jesus Gil Manzano wrote in his report that the Brazil international had "sarcastically applauded" the fourth official as he left the pitch.
The RFEF competition committee deemed that worthy of an additional two-game ban, meaning Neymar will miss league games against Real Sociedad, Madrid and Osasuna.