The warning came as American officials saw evidence that the country's embattled regime may be preparing to use such weapons to repel advances by rebel fighters.
"Today I want to make it absolutely clear to Assad and those under his command: the world is watching," Mr Obama said in a speech at the National Defence University in Washington.
"The use of chemical weapons is and would be totally unacceptable, and if you make the tragic mistake of using these weapons, there will be consequences and you will be held accountable."
US reconnaissance satellites and other tools last week detected increased activity at several chemical weapons depots in Syria, including movement of some weapons into position where President Assad's forces could more quickly deliver them by aircraft or artillery.
Intelligence sources also picked up orders to at least one Syrian military base to begin combining the two precursor chemical components of Sarin nerve gas to make it ready to use, officials said.
Syrian authorities have stored the components, which produce a deadly nerve agent when mixed, in secure separate facilities.
The Syrian foreign ministry yesterday denied the regime planned to use chemical weapons against it own people.
The 20-month uprising against Assad's minority Alawite ruling party – which the opposition Syrian Observatory for Human Rights said has killed more than 30,000 people – appears to be at or near a decisive moment, according to US officials.
Regime leaders may fear they may soon be forced to decide whether to flee into exile, fight to the death or delay defeat by using chemical weapons.
The fighting has drawn close to Damascus, the capital, and rebels fighting to overthrow the Assad regime have gained the ability to shoot down Syrian warplanes and helicopters with shoulder-fired anti-aircraft missiles supplied by Persian Gulf states.
The Syrian government yesterday sent fighter jets to bomb suburbs around Damascus to keep rebel fighters from the centre of the city, the Voice of America reported.
The US will take action if it sees evidence that the Syrian government is using chemical weapons, Secretary of State Hillary Clinton said yesterday. "This is a red line for the United States," she said in Prague. She declined to say what specific action the US might take.