"In my view, the project is in mortal danger," Oettinger told an event in Brussels on Tuesday night, according to Politico.
As well as outside opponents such as Russia and China, he said, "some within Europe want to weaken it or even destroy it – Poland, Hungary, Romania, the government of Italy."
Oettinger, a German, also criticized his own country's government for dragging its heels on the EU's next long-term budget, which he wants to see passed swiftly.
He has come into conflict with Italy before, especially after the new government threatened to cease contributions to the EU's budget over the bloc's rules on migrants arriving by boat across the Med.
Any such move by Rome, which would be the first time a member state had refused to meet its financial commitments, would result in interest charges and "possible further heavy sanctions", Oettinger warned last week.
Even before the Five Star Movement and the League formed their coalition government in June, Oettinger drew the ire of the two eurosceptic parties by telling an interviewer that he hoped the market turmoil that greeted Italy's election result would send "a signal to voters not to hand power to populists on the right and left".
Rome last month threatened to cut off its EU funding – just under €14 billion in 2016 – unless the rest of the bloc agrees to take in more of the people who continue to land on southern Italy's shores in the hope of migrating to Europe, saying that it doesn't want the "mickey taken out of us by the Union's other countries".
Since then EU ministers turned down Italy's request to redirect ships carrying migrants rescued at sea to other Mediterranean countries like France and Spain.
Italy is also heading for a clash with the EU over its national budget, with the coalition promising an end to years of austerity measures and Brussels insisting that Italy make a "significant effort" to control its huge public debt.
Speaking on Wednesday morning, Interior Minister and Deputy Prime Minister Matteo Salvini said that Italy would seek to stay within the limits imposed by the EU, while adding that "if we need to spend an extra billion to secure the country, we'll spend it".
ANALYSIS: Why Italy's standoff over migrants is a problem for the whole EU
Photo: Giovanni Isolino/AFP