Wary of interfering in Spain's domestic affairs, the EU representatives called for talks between the government in Madrid and Catalan authorities, but shied away from suggesting that the bloc could play a peacemaking role, despite appeals from Catalonia for European mediation.
Should Catalonia declare independence, Madrid would probably suspend the region's autonomy and take direct control of its government.
The stoppages, originally billed as a region-wide general strike but disavowed by the country's largest unions, affected the public sector, public transport and basic services.
Spain, which declared Sunday's referendum illegal and invalid, is bitterly opposed to any independence move.
The crisis in Spain grew more acute Sunday when some 900 people were treated in hospital following clashes with police ordered to prevent to the vote.
Mr Timmermans said the regional government of Catalonia had ignored the law by holding the referendum, and he said the matter was an internal one for Spain, which had to be dealt with in line with constitutional order of Spain.
"They have broken the democratic principles of the rule of law".
The Catalan government said the vote in support of independence was almost 90%, but official results have not yet been released.
He said Germany wasn't seeking to mediate in the dispute between Madrid and the regional government in Barcelona.
Top tourist attractions were also closed, including the city's famous Sagrada Familia church.
"I know very well that in Catalonia there is also much concern and great concern with the conduct of the autonomous authorities", he said.
King Felipe didn't mention the nearly 900 people who reportedly required medical care after Spanish police and security forces tried to prevent Catalans from voting Sunday, causing violent clashes.
On Tuesday, Spanish Interior Minister Juan Ignacio Zoido said: "We see how day after day the government of Catalonia is pushing the population to the abyss and inciting rebellion in the streets". But EU leaders have also warned about an escalation of violence and asked the Spanish and Catalan governments to hold a dialogue to reach a solution. A total of 90% of voters, or slightly more than 2 million people, said "yes" to Catalonia's independence.
Mr Puigdemont has long opposed the Madrid government's stance towards Catalonia and wants the European Union to assist with negotiations, even though the semi-autonomous region's longing glances outward have so far failed to garner robust support.
The vice president of the European Commission said Wednesday that Catalan leaders had disregarded the law, but that Madrid and Barcelona needed to negotiate a peaceful resolution.
Mr Puigdemont has called on the worldwide community to help mediate between the two sides.