Demonstrators waved the two-star flag of the Syria and pictures of President Bashar Assad outside the UN's office in the Mazzeh neighborhood of the Syrian capital.
Their placards read "Iraq will not happen again, this is Assad's Syria," and "Trump supports terrorism."
Some protesters chanted, "Death to America, death to Israel!"
US warships on Friday fired 59 Tomahawk missiles at a central Syrian air base from which Washington claims government jets launched a chemical attack that killed at least 87 civilians.
The Syrian government denied that it used toxic substances, saying it struck an arms depot used by extremists and describing the US strike as "foolish and irresponsible behavior."
"We came to denounce the American strike," said Ayyad Talab, head of the university students' branch in the National Syrian Students Union, which organized the protest.
"We want to say that we are ready to defend our country, armed with our work, our minds, and our ideas and with our souls if necessary," he told AFP.
The NSSU submitted a letter, addressed to UN Secretary General Antonio Guterres, that the United Nations mission in Damascus said it would forward to his office.
"We came to condemn the American aggression on our country. We consider it a disgrace to the democracy that America claims," protester Mahmoud Issa told AFP.