The Russian Investigative Committee said in a statement that the probe targets three Navalny associates -- Moscow city councillor Konstantin Yankauskas, and businessmen Nikolai Lyaskin and Vladimir Ashurkov -- accused of embezzling funds gathered for his campaign.
Navalny, the 37-year-old lawyer and anti-corruption blogger who became the charismatic leader of protests against President Vladimir Putin in 2011-12, is under house arrest while on trial in another fraud case.
The committee, which is the federal investigative authority equivalent to the FBI in the United States, said searches were being carried out in the homes of the three suspects.
Yankauskas told news agency ITAR-TASS he believed the investigation was an attempt to prevent him registering for Moscow city council elections in September.
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Quoting a source close to the investigation, news agency RIA-Novosti reported that searches were also taking place at the offices of Yandex.Dengi, an Internet payment portal associated with Russia's top search engine.
Navalny has faced a number of criminal cases his supporters have denounced as politically motivated.
In July last year, Navalny was convicted of embezzlement and sentenced to five years in jail but walked free the following day in a surprise move that allowed him to run for mayor. His sentence was later converted to a suspended one.