The Czech government decided on Monday that it would not accept a single refugee from the two countries.
Under the two-year EU programme in force since September 2015, the country of 10.6 million people has only accepted 12 refugees from Greece, against its quota of 2,600 out of a total of 160,000 refugees to be relocated.
"It saddens me that the Czech Republic has only relocated 12 people last year and none since," Juncker said in a speech at Charles University in Prague.
Despite steering largely clear of the Czech Republic, the recent migrant wave has become a hot political topic in the country facing parliamentary elections in October and presidential elections early next year.
In a February poll by the Czech Academy of Sciences, some 61 percent of respondents said the Czech Republic should not accept any refugees, while only three percent said it should accept them and let them settle down.
Thirty-two percent said they would temporarily accept refugees but would want them to return once things calmed down in their countries of origin.
Juncker said he was "trying to understand the national sensitivities", but he urged the Czechs to show more solidarity.
"For me the Czech Republic and the Czechs are very European. So it must be European in migration too."
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