Barack Obama said he had “at this point not ordered military engagement”.
But he added: “There would be enormous consequences if we start seeing movement on the chemical weapons front or the use of chemical weapons.”
Earlier the new UN special envoy to Syria faced criticism for refusing to say whether President Bashar al-Assad must quit.
Barack Obama, speaking to reporters at a White House briefing, said the deployment or use of biological weapons would widen the conflict in the region.
He said: “It doesn’t just include Syria. It would concern allies in the region, including Israel, and it would concern us.”
He warned President Bashar al-Assad and “other players on the ground” about the use or movement of such weapons.
He said: “A red line for us is [if] we see a whole bunch of chemical weapons moving around, or being utilized. That would change my calculus.”
Syria holds the world’s fourth-largest stockpile of chemical weapons. Last month a Syrian foreign ministry spokesman said the weapons would never be deployed inside Syria.
However, the US has seen unconfirmed reports recently that the Syrian authorities have been moving the country’s chemical arms stockpile.
Fighting continued in several Syrian cities on Monday, including Damascus, Deraa and Aleppo.
A Japanese journalist, Mika Yamamoto, was killed by gunfire in Aleppo, the country’s foreign ministry has confirmed.
Mika Yamamoto, 45, was a veteran war reporter, working for Japan Press.
The UN says more than 18,000 people have been killed in the conflict, 170,000 have fled Syria and 2.5 million need aid within the country.
Earlier on Monday, the UN’s new envoy to Syria, Lakhdar Brahimi said he was “not in a position to say yet” whether President Assad should go, but was “committed to finding a solution”.
Lakhdar Brahimi, a former Algerian foreign minister, last week succeeded Kofi Annan who resigned after both sides largely ignored his peace plan.
On Sunday, UN observers ended their mission to verify its implementation.
Their departure came after the UN Security Council agreed to allow their mandate to expire at midnight, and instead set up a new civilian office in Damascus to pursue political contacts that might lead to peace.
Since being confirmed as the new UN and Arab League envoy to Syria, Lakhdar Brahimi has acknowledged that he has no concrete ideas of how to end the conflict, which he believes has been a civil war for some time.
On Monday, he said he was not ready to say whether President Assad should step down despite widespread international condemnation of his government’s crackdown on dissent since protests erupted in March 2011.
“I am not in a position to say yet, because I was appointed a couple of days ago. I am going to New York for the first time to see the people who I am going to work for, and I am going to Cairo see the Arab League,” he explained.
After announcing his resignation, Lakhdar Brahimi’s predecessor, Kofi Annan, said: “It is clear that President Bashar al-Assad must leave office.”
The main opposition coalition, the Syrian National Council (SNC), said Lakhdar Brahimi’s stance showed “disregard for the blood of the Syrian people and their right of self-determination” and demanded he apologize.
Lakhdar Brahimi stressed that he was “committed to finding a solution full stop”.
“I am a mediator. I haven’t joined any Syrian party. I am a mediator and a mediator has to speak to anybody and everybody without influence or interest,” he added.
“Then I’ll make up my mind about what to say and what to do.”
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