MIAMI — Two fugitive brothers from Ecuador, whose relatives have contributed hundreds of thousands of dollars to American political campaigns, are being investigated by the Department of Homeland Security to determine whether any of the millions the men were accused of pilfering in their home country made it to the United States, according to a federal law enforcement official familiar with the case.

Two years ago in Ecuador, the brothers, Roberto and William Isaias, were sentenced in absentia to eight years in prison for embezzling millions from a bank they owned there. In South Florida, where they now live, many see them as respected businessmen, and their family members have donated more than $300,000 to the political campaigns of Democrats and Republicans, including $90,000 to President Obama’s re-election effort in 2012.

Last year, Washington denied Ecuador’s request to extradite the brothers, saying the Ecuadorean government lacked sufficient proof. Ecuador sent its attorney general to Washington this month to urge its embassy to press the Isaias matter.

The issue has damaged already difficult relations between the two countries, which have been at odds over two other international fugitives: Julian Assange, the WikiLeaks founder taking refuge in the Ecuadorean Embassy in London, and Edward J. Snowden, the former intelligence contractor to whom Ecuador also showed an openness to granting asylum.