The Brussels boss said there can be no mediation in Spain when only one-sided asked for the EU’s independent help to solve the crisis.
And he warned the split could encourage other regions to do the same and make governing the EU too complicated.
In a speech at Luxembourg University, he said: “If we allow Catalonia – and it is none of our business – to separate, others will do the same. I do not want that.”
He said he was “very worried” about separatist tendencies in Europe and had encouraged Spanish Prime Minister Mariano Rajoy to ensure the situation was brought under control.
Referring to Catalan calls for EU mediation, he said the Commission could not mediate if only one side asked it to do so. The EU has said it has confidence in Rajoy to resolve what it sees as an internal issue.
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And he revealed his concerns that the referendum result could tear the European Union apart as the crisis in Spain deepens.
But the President of the European Commission seemed a little of stressed than he was letting on following his dramatic speech.
Taking questions from students, the EU chief mopped his brow with a napkin and later wiped his face before the Q&A begun.
Violence broke out in Spain on Thursday as independence campaigners clashed with pro-unity protesters during the country’s National Day.
The groups were seen hurling metal chairs and tables at one another in Barcelona before police moved in to break up the violence.
Families with Spanish flags were seen fleeing as chairs were used as missiles when the two camps faced off.
Catalan government chief Carles Puigdemont had stopped short of moving a motion of independence in the regional parliament, disappointing many of his own supporters.
But Prime Minister Mariano Rajoy has given the Catalan leader five days to clarify whether or not he has declared independence.
If Mr Puigdemont does not respond in time or confirms a will to secede, Mr Rajoy can turn to article 155 of the 1978 constitution which allows him to sack the regional government.
Ahead of the National Day, Mr Puigdemont tweeted: ”Ask for dialogue and they answer you by putting the 155 on the table. Understood.”
But Mr Rajoy warned: “There is an urgent need to put an end to the situation that Catalonia is going through – to return it to safety, tranquillity and calm and to do that as quickly as possible.”