Foreign Minister Srdjan Kerim said that the PDP's refusal to take part amounted to "self-isolation" and added that the delegation was nonetheless "very representative".
It puts Macedonia on the path towards eventual EU membership, ahead of all other Balkan states.
EU officials want the agreement to stimulate talks designed to address the grievances of the minority ethnic Albanian community, but in a sign of continuing distrust, one Albanian party - the Party for Democratic Prosperity (PDP) - refused to join the delegation.
Results promised
In talks with EU foreign ministers, Macedonian leaders said the talks would achieve concrete results by June.
Foreign Minister Srdjan Kerim said that the PDP's refusal to take part amounted to "self-isolation" and added that the delegation was nonetheless "very representative".
The Macedonian Government includes members of another ethnic Albanian party, the Democratic Party of Albanians.
Prime Minister Ljubco Georgievski hailed the agreement.
"This agreement is recognition of all that we have achieved in the last 10 years in the fields of democracy, multi-ethnic cohabitation, reforms, regional and international co-operation," he said.
National groups
"At the same time it is an obligation to achieve even more in all these fields in the future."
Javier Solana, the EU's chief of foreign and security policy, called the signing "an important opportunity to achieve real progress in moving forward the process of enhanced dialogue".
The BBC's Central Europe correspondent, Nick Thorpe, says that accelerating the country's approach to the EU could help the different national groups living in the country to forget their disagreements.
But diplomats say that if Macedonia hands over a letter formally asking to join the EU, there would have to be a long review before negotiations could even begin.
Albanian demands
Albanians are believed to make up about one third of the population and are demanding the status of an equal nation, not simply a minority in the country alongside the far smaller communities of Turks, Vlachs and others.
Mr Kerim said he did not exclude the possibility of political dialogue with Albanian leaders leading to constitutional change.
"It depends on all political parties," he said.
Three rounds of talks have already been held in Skopje under the chairmanship of President Boris Trajkovski in the search for a Macedonian-Albanian compromise.