The Czech Republic has not relocated anyone since August 2016 and not made any new pledges for over a year.
The commission, the EU's executive body, accused the three countries of "non-compliance with their legal obligations on relocation".
The European Commission has announced it will sue the Czech Republic, Poland and Hungary in the bloc's top court for their refusal to take in asylum-seekers in line with the EC's mandatory redistribution mechanism. The plan involved the relocation of 160,000 people.
The Commission has launched legal action against the three member states earlier this year, sending them a "Letter of Formal Notice" in June and a "Reasoned Opinion" in July.
The Commission also said it would take Hungary to the ECJ, the EU's highest court, over its controversial higher education law, which critics say curbs academic freedom.
Czech Prime Minister Andrej Babis told the BBC he opposed the relocation plan and that it fueled anti-migrant sentiment in the country.
The Commission said Thursday that the three "remain in breach of their legal obligations" and "have given no indication that they will contribute to the implementation of the relocation decision".
Reacting to the EC's latest decision at a press conference in Brussels, Minister of Foreign Affairs and Trade Péter Szijjártó said the mandatory resettlement quotas are impossible to implement, unreasonable and a violation of European Union rules, and vowed that the government will defend its position before the CJEU.
Hungary's right-wing government is looking to pass a higher education law that could close the Central European University, founded by financier and philanthropist George Soros.
Hungary also caused controversy in June when it passed legislation forcing non-governmental organisations to declare themselves "foreign-funded".