Neymar's mother Nadine Goncalves and the family company N&N also had appeals rejected.
"Santos FC, Barcelona FC, Neymar, his mother Nadine Goncalves and N&N, the family company, have lost their appeals to have charges of fraud and corruption dropped," the High Court said in a statement.
They cannot appeal against the Court's decision.
The case stems from a complaint by Brazilian investment group DIS, which owned part of Neymar's transfer rights and alleges that it received less money than it was entitled to when Neymar joined Barcelona from Santos in 2013.
Investigations were carried out in Spain and Brazil into whether any part of Neymar's transfer fee had been concealed when he moved to Barcelona from Santos.