The former Bosnian Serb military commander has been charged with genocide by a UN war crimes tribunal in the Hague over the 1995 Srbrenica massacre.
"Unless Ratko Mladic is in the Hague by the end of the month, then we will have no other option but to disrupt the negotiations," said EU Enlargement Commissioner, Olli Rehn.
Zoran Stojkovic, the Serbian justice minister, said Belgrade was "working intensely" to find Mladic.
"We must find the individual we are looking for so that we can fulfil our commitment to The Hague tribunal," Stojkovic said.
If Belgrade fails to deliver Mladic by Monday, talks on a pre-membership agreement that had been scheduled for May 11 would be called off, Rehn said.
The agreement would offer Serbia trade privileges and closer political ties.
The commissioner said he would meet UN war crimes prosecutor Carla Del Ponte next Wednesday to discuss the case.
Vojislav Kostunica, Serbia's prime minister, has pledged to deliver Mladic to the Hague tribunal.
UN prosecutors say Mladic is hiding in Serbia with the help of military hardliners.
Mladic and former Bosnian Serb political leader Radovan Kardazic are wanted over the genocide of 8,000 Muslims at Srebrenica – the worst mass killing in Europe since the end of the second world war.