The mayor of La Linea de la Concepcion took the decision even though the Congolese team has been preparing for the tournament in Belgium and almost all of its players and staff are based in Europe.
Congo play a friendly match in Lidge on Wednesday against Denmark and had planned a second preparatory game in Spain next Monday before heading off to the World Cup, co-hosted by Canada, Mexico, and the United States.
But on Tuesday, La Linea’s mayor, Juan Franco, revoked authorisation for the match.
"The health situation in the Democratic Republic of the Congo regarding the Ebola virus, together with the documentation that has been provided to us, which does not fully demonstrate that there is no risk whatsoever, means that this is the most prudent decision," a statement released by his office said.
The Congolese Football Federation (FECOFA) said on Wednesday that the team was in compliance with all health, sporting and regulatory requirements. It said it was in discussions with the relevant authorities and the Spanish Football Federation to find a solution that would allow the match to go forward.
The decision by the mayor caught Congo coach Sebastien Desabre by surprise when he was asked about it at a press conference on Tuesday.
"I'm sure a solution will be found," he said, suggesting the possibility of playing the match at a different stadium in Spain.
"The DR Congo national team respects the decision, which is based on fears of Ebola. We speak with FIFA every day about Ebola. Our doctors are constantly in contact with FIFA, and we strictly follow the health protocols."
The Congolese team, appearing at their first World Cup in 52 years, are to be based in Houston. They open their Group K campaign against Portugal on June 17th, before facing Colombia in Guadalajara on June 23rd and Uzbekistan in Atlanta on June 27th.