Usmanov is personally suing Navalny and his Anti-Corruption Foundation which produced the report.
Usmanov has launched action against the Kremlin critic for the "protection of his honour and dignity" at Moscow's Lyublinsky court, the court's press service told TASS state news agency.
Navalny, who has announced he intends to stand for president in 2018, in March released a report claiming Medvedev controls a property empire through shadowy not-for-profit organisations. The video has been viewed more than 18 million times on YouTube.
Medvedev has denied the claims, which he described as "malarkey."
Navalny's supporters last month held the largest unauthorised demonstration in Moscow in years, protesting over the accusations against the prime minister, one of President Vladimir Putin's closest associates.
Usmanov was listed as Russia's third richest businessman by Forbes magazine in 2016 with a fortune of $12.5 billion. His interests range from metallurgy to social networking sites Mail.ru and VK and business daily Kommersant.
Navalny's report says Usmanov donated a mansion in an elite suburb of Moscow whose value it estimates at $70 million to a foundation it links to Medvedev.
The report says: "We know very well what such a gift means: it is a bribe."
Usmanov is personally suing Navalny and his Anti-Corruption Foundation which produced the report, according to the website of Moscow city courts.
In an interview with Vedomosti independent business daily on Wednesday, Usmanov said he gave the house and grounds to the foundation in exchange for land it owned, which he bought at a "nominal price".
He said the house and the land were worth approximately the same -- around $50 million.
"This is no bribe!" Usmanov told Vedomosti.
He said he planned to sue Navalny "over the lying" in the report.
Navalny, who is a lawyer, responded defiantly on Twitter, saying he would call Medvedev as a witness.
He rejected Usmanov's description of the deal, saying the billionaire bought the land from commercial companies.
Navalny has been targeted in a number of legal cases he describes as politically motivated.
This year he was convicted of fraud in a retrial after the European Court of Human Rights quashed the original verdict.
This conviction would bar him from standing for public office.