The two men, one from government forces and one from the rebels, were "responsible for perpetrating unthinkable violence against civilians," US Secretary of State John Kerry told reporters.
The move against Marial Chanuong, also known as Marial Chinoum, a commander of the South Sudanese presidential forces, and Peter Gadet, a leader of the anti-government forces, comes only days after Kerry visited Juba and called for both sides to lay down their arms.
"We will do our utmost to prevent South Sudan from plunging back towards violence and despair," Kerry said after meeting EU foreign policy chief Catherine Ashton in Washington.
"We will hold accountable those who have stood in the way of a peace plan."
The sanctions move will be a tough one for Kerry since Washington was the prime mover in enabling South Sudan to split from Sudan and win independence in 2011.
"We expect them to serve as a warning to those engaged in the cycle of violence," a senior administration official told reporters on a conference call.
The sanctions will freeze any assets the two men may have in the United States and prevent US companies from having any dealings with them, while they will also be slapped with a visa ban.
UN chief Ban Ki-moon visited Juba on Tuesday as rebels and government forces battled for control of a key oil town, in the latest major drive for a ceasefire in a war that has seen the nation collapse amid a brutal cycle of war crimes.
Chanuong was accused of leading violent attacks against civilians in Juba in December 2013 when the violence first erupted as President Salva Kiir accused his former vice president Riek Machar of leading a coup attempt.
Gadet was said to be behind an attack in which 200 people died in Bentiu on April 17.
"We will continue to stand with the people of South Sudan who called for peace and who recognized that the only way to resolve this conflict is through a political dialogue," Kerry said.
The UN chief said South Sudan rebel chief Machar had promised to attend fresh peace talks with Kiir tentatively scheduled to take place on Friday in Ethiopia.
The two men are from the two main tribes in South Sudan, the Nuer and the Dinka, and what was seen initially as a power struggle has erupted into a wider, bloody ethnic conflict.
Ashton warned the EU was also considering imposing sanctions, adding she feared that Sudan was "on the brink of what could be a civil war, ethnically motivated."
"The prospects for famine and a humanitarian disaster, they're really looming large now. So we need to work together" to ensure the country's leaders come to the negotiating table, she added.
