On Thursday, the jury in the trial unanimously concluded that prosecutors had failed to prove the defendants' guilt and all three were released in the courtroom.
"Since the jury agreed to acquit [the defendants], I will bring a not guilty verdict and the case must be referred back to the investigators," Judge Yevgeny Zubov said.
Public prosecutor Yulia Safina said earlier that the prosecution would appeal the jury's decision.
Novaya Gazeta reporter Politkovskaya, who gained international recognition for her reports of military atrocities against civilians in the troubled Caucasus republic of Chechnya, was gunned down in an elevator in her Moscow apartment building in October 2006, in what police described as a contract killing.
Dzhabrail and Ibragim Makhmudov, brothers from Chechnya, and former police officer Sergei Khadzhikurbanov were charged with involvement in the murder, but the man suspected of pulling the trigger, Rustam Makhmudov - the eldest of the three Makhmudov brothers - remains at large. A separate case has been opened against him.
Lawyers for Politkovskaya's relatives also said they would seek another investigation.
"We want the real killer [in the dock], and we will succeed," lawyer Karina Moskalenko said.
She declined to give an opinion on the verdict, saying it was "neither good nor bad."