Slovenia has introduced new border restrictions for migrants as part of efforts to close the Balkans route from Greece to Western Europe.
Only migrants who plan to seek asylum in the country, or those with clear humanitarian needs will be allowed entry.
In reaction, Serbia said it would close its borders with Macedonia and Bulgaria to those without valid documents.
The future of the EU’s passport-free Schengen zone is already in doubt.
Eight of its members, including Austria, Hungary and Slovakia, have tightened border controls, leaving thousands of migrants stranded in Greece.
Europe is facing its biggest refugee crisis since World War Two. Last year, more than a million people entered the EU illegally by boat. Most of them were Syrian, fleeing the country’s civil war.
Slovenia, which is a EU member, has been used as a transit country by migrants trying to reach Germany and other northern European states.
But Slovenian Prime Minister Miro Cerar said on Tuesday the Balkan route was now effectively “shutting down”.
He said the restrictions, which came into force at midnight local time (2300 GMT), were part of a wider initiative which would see other Balkan countries, as well as Greece and with the cooperation of Turkey, turn back “all irregular migrants”.
The EU and Turkey are considering a radical plan including proposals to return all migrants arriving in Greece sent back to Turkey. For each Syrian sent back, a Syrian in Turkey would be resettled in the EU.
The UN expressed concern at the plan on Tuesday, while Amnesty International called it a death blow to the right to seek asylum.
Speaking to the BBC, Thorbjorn Jagland, Secretary General of the Council of Europe, said the proposal to send migrants back would contravene international law.
The deal, discussed at a summit in Brussels on Monday, has not been finalised and talks will continue ahead of an EU meeting on 17-18 March.
European leaders are billing their new proposal to deal with the refugee and migrant influx as a “game-changer”, but the scheme is not agreed yet and there are doubts about whether it it is practical or even legal.
The centrepiece is a plan to take any refugees and migrants who cross the sea to Greece in smugglers’ boats and return them, directly, to Turkey.
EU officials say whatever is finally agreed “will comply with both European and international law”. Privately, though, some admit that, while the assessment of their lawyers is “quite promising”, there are legal hurdles that must be overcome.