The move raises official pressure on the charismatic 37-year old lawyer, who has emerged as the most popular opposition figure. It will also add to international concerns about selective, politically motivated justice in Russia.
The Investigative Committee said in a statement Tuesday that Navalny and his brother Oleg are accused of stealing more than 26 million rubles (more than $810,000) from a branch of French cosmetics company Yves Rocher by charging an excessive price for shipping services.
Navalny scoffed at the charges.
"In a sense, it's good news. It shows that we are doing the right thing and they are afraid of us," he said on his blog.
In July, Navalny was convicted of embezzlement in a separate case, but a court suspended his five-year sentence earlier this month. Navalny won 27 percent of Moscow's mayoral vote in September, finishing a strong second behind the Kremlin-backed incumbent and cementing his positions as Russia's No. 1 opposition leader.
Under the Russian law, the conviction bars Navalny from running for political office for life, but he has shown no sign of slowing down his political activities.
He recently has sought to raise the heat on authorities by launching a campaign to revise utility fees ahead of the city council election next year.
Navalny first made a name for himself with investigations of official corruption on his blog, which quickly became an Internet sensation. He currently has more than 400,000 followers on Twitter.
In the winter 2011-2012, his energy and ambition made him a driving force behind a series of opposition protests Vladimir Putin's return to the Russian presidency, which drew 100,000 or more to the streets of Moscow.