The espionage is "incompatible with democratic co-existence between friendly countries", Dilma Rousseff said in a statement Monday.
The US National Security Agency (NSA) spied on Petrobras, Brazil's TV Globo reported Sunday, citing documents from whistleblower Edward Snowden.
The network's flagship news magazine, "Fantastico", showed an NSA training slide with the heading, "Many targets use private networks".
Targets listed on the slide include Petrobras, Google, the French foreign ministry, and SWIFT, a provider of secure financial messaging services to institutions worldwide.
The appearance of Petrobras among surveillance targets "contradicts NSA's affirmation that the spying does not have economic or commercial objectives", TV Globo said.
The disclosure came a week after TV Globo said the NSA intercepted telephone calls and e-mails of Rousseff and Mexican President Enrique Peña Nieto.
Last week's report was also based on documentation from Snowden, a former NSA contractor who has been working with Brazil-based US journalist Glenn Greenwald.
"There is no doubt that Petrobras represents no threat to the security of any country," Rousseff said Monday.
"What it does represent is one of the largest petroleum assets in the world and the patrimony of the Brazilian people," she added.
Besides demanding an explanation, Brazil will insist the US government take "concrete steps that definitively exclude the possibility of espionage which violates human rights, our sovereignty and our economic interests", she said.