Speaking to reporters at the Pentagon, Defense Secretary Jim Mattis said the attack was necessitated "by offensive movement with offensive capability of what we believe were . . . Iranian-directed forces inside an established and agreed-upon deconfliction zone."
If confirmed, it would be the first U.S. strike on Iranian proxies during seven years of war in Syria, signaling a possible escalation against Assad's most important ally.
Under the Obama administration, U.S. forces steered clear of direct confrontations with Iran and its proxies across Syria, Iraq and Yemen, prioritizing the upholding of Washington's nuclear deal with Tehran above all else.
"If U.S. troops are now engaging directly with Iranian militias, escalation in the absence of a well-wrought plan could inflame the conflict in Syria and further afield," Thanassis Cambanis, a fellow at the Washington-based Century Foundation, wrote in a research note published Friday.
Rebel commanders said the convoy, made up of Syrian and Iraqi militiamen who now form the bulk of Assad's fighting force, retreated after the U.S. attack and had not attempted to advance again by sunset Friday.
The airstrike comes at a time when the U.S. presence in Syria has become increasingly visible. Last month, the Trump administration launched the first direct U.S. attack on Assad targets, striking an air base in the central province of Homs in retaliation for a chemical attack that killed scores of civilians in the country's northwest. On the ground, the U.S. presence in Syria is also becoming more visible, as American troops back Kurdish-led forces fighting the Islamic State.
Aerial bombardments have ebbed across Syria since early May, after Russia, Iran and Turkey backed a deal to freeze fighting between government and rebel forces across four "de-escalation zones."
In areas outside of that pact, the cease-fires have freed up resources on all sides and triggered a scramble for influence around the fringes of Islamic State-held territory in eastern Syria.
The area is home to lucrative energy and agricultural sites that will be vital to Assad's government if it is to win economic independence from its Russian and Iranian backers.
Control of eastern Syria also includes the power to block or link up Iranian supply routes from Tehran to its most important proxy, Hezbollah, in Lebanon.
"Regime-allied militias advanced from the west and got bombed when they tried to enter our area. They have been advancing in recent weeks, bombing us heavily as they came," said Col. Muhannad al-Talla, a commander of Maghawir al-Thawrah, a Pentagon-backed rebel group at the al-Tanf air base.
Syrian officials bridled at Thursday's airstrike. On state television, an unnamed government official described it as a "flagrant aggression" by the "American-Zionist project in the region."
In Geneva, a fresh round of talks between Syrian government and opposition officials made little headway this week as the two sides refused to meet in the same room and none of the agenda's main issues was discussed.
Missy Ryan in Washington and Zakaria Zakaria in Istanbul contributed to this report.