About 6000 people took part on Monday in different marches that converged on the historical central part of Guatemala City where the National Congress is located. Congress approved the treaty on Thursday.
The protests were part of a national one-day strike against the treaty.
Activists shouted slogans against the United States and against ruling party legislators. Other activists gathered outside the US Embassy, and yet another group marched on a highway to the Atlantic.
There were similar protests across Guatemala with farmers blocking highways leading to the borders with Mexico, El Salvador and Honduras.
Guatemalan President Oscar Berger played down the protests and warned that if blockades were set up, leaders would be arrested.
He said CAFTA would be beneficial for Guatemala and especially would bring in foreign investment.
"When our producers get big dollars, we'll see if they stage a demonstration," he said.
Interior Minister Carlos Vielman warned that if blockades were not withdrawn voluntarily, they would be forcibly dispersed.
The Guatemalan Congress approved the seven-nation CAFTA by a 126-12 vote, making Guatemala the third Central American country to ratify the treaty after Honduras and El Salvador.
Costa Rica, Nicaragua, the Dominican Republic and the United States have yet to ratify it.