Gen Mladic has been indicted over the 1995 Srebrenica massacre
"It is now high time to... locate, arrest and transfer Ratko Mladic to the Hague (tribunal) without delay," said EU Enlargement Commissioner Olli Rehn.
The EU has given Belgrade until Sunday to arrest General Mladic.
The former Bosnian Serb military chief is charged with genocide over the 1995 Srebrenica massacre and other crimes.
Last month, the EU decided to carry on with the talks after assurances by UN Chief War Crimes Prosecutor Carla del Ponte and Serbian Prime Minister Vojislav Kostunica that Gen Mladic would be arrested.
For Serbia-Montenegro, this is the last chance to keep its European hopes alive, the BBC Oana Lungescu in Brussels says.
'No other option'
Mr Rehn's remarks came in Brussels, following his talks with Serbia-Montenegro's Foreign Minister, Vuk Draskovic.
Mr Rehn told a joint news conference that he appreciated Belgrade's recent actions to arrest five alleged aides to General Mladic.
But he added that the EU had "no other option than to call off the next round of negotiations" if Gen Mladic was not arrested within days.
The talks are planned for 11 May, and Mr Rehn said he would meet Mrs del Ponte next Wednesday.
But barring last-minute surprises, her report is bound to be negative, our correspondent says.Now, the EU has to stick to its word or lose credibility, she adds. BBC