Jan Fischer (left) says he will take Mr Klaus' demand to EU leaders
The Czech Republic should sign the Lisbon Treaty and not erect "artificial obstacles" to ratification, says EU Commission chief Jose Manuel Barroso.
Mr Barroso was speaking after meeting Czech Prime Minister Jan Fischer in Brussels.
The Czech Republic is the only EU member still to ratify the treaty, which is meant to reform the bloc.
Eurosceptic Czech President Vaclav Klaus last week demanded fresh concessions before he would sign it.
Mr Klaus demanded an opt-out from the Charter of Fundamental Rights, similar to opt-outs agreed previously for the UK and Poland.
Mr Klaus raised fears about possible property claims by Germans expelled from the then Czechoslovakia after World War II.
But Mr Barroso said: "We certainly hope that no artificial obstacles are raised at this time.
"We expect the Czech Republic to honour the commitment it has taken. It is in the interests of nobody, least of all the interests of the Czech Republic, to delay matters further.
"Good faith and loyal co-operation are principles of European law and international law."
Since the Irish people voted to support the treaty in a referendum earlier this month, the spotlight has turned on the Czech Republic.
Even though the government supports the treaty and parliament has ratified it, Mr Klaus is holding out.
However, he could not sign it right now even if he wanted to, since the country's Constitutional Court is considering a legal challenge mounted by Eurosceptic senators.
The court announced on Tuesday that it would hold a hearing on the complaint on 27 October.
Prime Minister Jan Fischer has said he will take Mr Klaus' demand for an opt-out to European leaders when they meet in Brussels on 29 October - provided Mr Klaus guarantees he will then sign the treaty without delay.