The CAS refused to allow the request for a temporary suspension of a FIFA sanction.
In January, football's world governing body had ordered that they play the Group D match on March 22 in an empty stadium.
FIFA's disciplinary committee found a group of Hungary supporters had chanted anti-Semitic abuse and displayed offensive symbols in their friendly against Israel in Budapest last August.
Hungary and Romania stand second and third in the group respectively, with both teams having nine points, three fewer than leaders Holland.
The Hungarian Football Federation (MLSZ) have appealed to the Court of Arbitration for Sport (CAS) to stay the decision and the arbitration process is ongoing.
CAS announced on Tuesday: "The Court of Arbitration for Sport has dismissed the application for provisional measures filed by the Hungarian Football Federation in relation to a decision of the FIFA instances ordering that the Hungarian team play its next home match in the preliminary round for the 2014 FIFA World Cup without spectators."
The CAS statement added: "The HFF had requested the CAS to stay the effects of the decision concerning the match to be played without spectators, a request which was denied by CAS today. As a consequence, the decision imposing that the match between Hungary and Romania on 22 March 2013 be played without spectators is maintained.
"The arbitration in this matter is ongoing and an award on the merits will be issued at the end of the CAS procedure."