The response came after the US State Department announced on Tuesday it would deny visas to five European nationals, accusing them of seeking to "coerce" American social media platforms into censoring viewpoints they oppose.
France, Germany and Spain also condemned the news from Washington.
A statement from the Commission said: "We have requested clarifications from the US authorities and remain engaged. If needed, we will respond swiftly and decisively to defend our regulatory autonomy against unjustified measures.
"Our digital rules ensure a safe, fair, and level playing field for all companies, applied fairly and without discrimination," it added.
Breton, the former top tech regulator at the European Commission, often clashed with tech tycoons such as Elon Musk over their obligations to follow EU rules.
The State Department has described him as the "mastermind" of the European Union's Digital Services Act (DSA), which imposes content moderation and other standards on major social media platforms operating in Europe.
The DSA stipulates that major platforms must explain content-moderation decisions, provide transparency for users and ensure researchers can carry out essential work, such as understanding how much children are exposed to dangerous content.
But the act has become a bitter rallying point for US conservatives who see it as a weapon of censorship against right-wing thought in Europe and beyond – an accusation the EU furiously denies.
"The Trump Administration will no longer tolerate these egregious acts of extraterritorial censorship," US Secretary of State Marco Rubio said in a post on X Tuesday.
'Intimidation and coercion'
German Foreign Minister Johann Wadephul wrote in a post on X Wednesday: "The DSA was democratically adopted by the EU for the EU –- it does not have extraterritorial effect.
The visa bans, he added, "are not acceptable".
French President Emmanuel Macron said on X: "France condemns the visa restriction measures taken by the United States against Thierry Breton and four other European figures."
"These measures amount to intimidation and coercion aimed at undermining European digital sovereignty," he added, saying Europe would defend its "regulatory autonomy".
Spain's foreign ministry also condemned the US sanctions, saying that "a safe digital space, free from illegal content and disinformation, is a fundamental value for democracy in Europe and a responsibility for everyone."
Breton left the commission in 2024 and Stephane Sejourne, his successor in charge of the EU's internal market, said on X that "no sanction will silence the sovereignty of the European peoples".
The visa ban also targeted Imran Ahmed, from the Center for Countering Digital Hate, as well as Anna-Lena von Hodenberg and Josephine Ballon of HateAid, a German organisation that the State Department said functions as a trusted flagger for enforcing the DSA.
Clare Melford, who leads the UK-based Global Disinformation Index (GDI), was also on the list.
A statement from HateAid called the US government decision an "act of repression by an administration that increasingly disregards the rule of law and tries to silence its critics with all its might".
A GDI spokesperson said that "the visa sanctions announced today are an authoritarian attack on free speech and an egregious act of government censorship."
It called the actions "immoral, unlawful, and un-American".
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