"The court rejects the appeals of the defendants and confirms the death penalty," Judge Fathi Dahn said after a five-minute hearing.
The five Bulgarian nurses and the Palestinian doctor were found guilty late last year of intentional infecting more than 400 Libyan children with HIV in the late 1990s. All worked in the Al-Fateh Children's Hospital in the city of Benghazi.
The defendants have been incarcerated since 1999. They deny intentionally infecting the children and claim that they were tortured while in Libyan prisons, saying the confessions at the center of the prosecution's case were coerced.
In 2003, a French doctor charged with investigating the outbreak of HIV at the hospital found that it had started before the six medics arrived there.
The sentence has become an international political issue, with Bulgaria, the EU and the US urging Libya not to carry out the executions.
"We regret that these decisions have been taken, but I would also like to express my confidence that a solution will be found," EU Commission President Jose Manuel Barroso told the European Parliament after the Libyan court's ruling was announced.
The case now goes to the government-controlled High Judicial Council, which has the authority to change the sentence or even pardon the defendants. The Council will meet on July 16.
"We hope things will be clear on Monday," Bulgaria's Prime Minister Sergei Stanishev said on Bulgarian radio. "The decision of the Council will open the way for solving the case on a political level and will be the final step in the talks held in the last few months."
There has been speculation that the six medics could be freed in exchange for money to provide for the infected children's medical care.
On Tuesday, it was reported that such an agreement had been mediated by the Gadhafi Foundation, a charity organization run by one of the sons of Libyan leader Moammar Gadhafi.
But the families of the children have reportedly asked for 10 million euros ($13.7 million) in compensation per child, a sum the EU has been unwilling to pay. Bulgaria also rejects the term "compensation," saying that the word implies the medics are guilty.