The much-anticipated petition, whose authors include a founder of Rousseff's Workers' Party, was handed over to congressional lower house speaker Eduardo Cunha.
Cunha must now decide whether to submit the petition to Congress, which would possibly trigger an impeachment trial.
"We are confident that the lower house speaker (Cunha) now has all the elements he needs to accept an impeachment process, which is not just a request from the opposition - it's something Brazil is asking for," said Carlos Sampaio, leader of the opposition PSDB in the lower house.
"We will evaluate it," the speaker said.
Rousseff, re-elected in October 2014, is accused of taking unauthorized state bank loans to cover budget holes both ahead of and after the start of her second term.
The nation's accounting court gave the opposition ammunition earlier this month when it partially backed the allegations.
The latest of several petitions filed with Cunha is seen as carrying more weight than earlier versions.
The authors include Helio Bicudo, a Workers' Party veteran who has become a sharp critic of Rousseff. It is supported by the main opposition PSDB and several other parties.
However, Rousseff says that the accounting irregularities were nothing more than a common method used by governments for years to shuffle their budgets, and describes the impeachment push as "a coup."
Bicudo shot back today, saying that the country has turned on Rousseff.
"We have to go onto the streets to fight for this ideal, for a better Brazil," Bicudo told journalists.
Kim Kataguiri, leader of opposition group Movimento Brasil Livre, said activists would camp outside Congress "so they accept the impeachment request."
Some analysts believe he may wait to see the result of an appeal against a Supreme Court decision on the rules of how the impeachment procedure can go ahead.
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