The US-led coalition is supporting an alliance of Kurdish and Arab fighters in their assault on ISIL in its de -acto capital of Raqqa in northern Syria.
The Britain-based observatory says at least 15 IS jailers and fighters were also killed in the strike in the Deir El-Zour province.
Pentagon spokesman Adrian Rankine-Galloway confirmed coalition strikes on Mayadeen on Sunday and Monday, targeting IS "command and control facilities" and other "infrastructure".
"If we are responsible for any civilian casualties we come forth and admit it", he said.
Most of Deir Ezzor province is controlled by the militants and it has been the target of air strikes by both the coalition and the Syrian army and its Russian ally.
Describing American reassurances to Turkey that the USA will take back weapons given to the Syrian Kurds after the ISIS fight, Mattis said it depends on when or where the next mission is.
Mattis also said that as the fight against ISIS grows more complex and moves into the Euphrates River Valley, it will require more deconfliction with the Russians.
"If, however, Mr Assad conducts another mass murder attack using chemical weapons, he and his military will pay a heavy price".
Observatory head Rami Abdel Rahman said 222 civilians, including 84 children, were killed in the largely Islamic State group-held province of Deir Ezzor.
The regime denied any use of chemical weapons.
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Pentagon spokesman Navy Captain Jeff Davis said Tuesday the latest USA warning was the result of activity at the same airbase "that indicated preparations for possible use of chemical weapons".
Russian President Vladimir Putin's spokesman Dmitry Peskov says that "such threats to Syria's legitimate leaders are unacceptable". While the Pentagon has eyed the region as the next place to attack the militants, Syrian government forces and Iranian-backed militias have also made a concerted effort to move into the area.
Iran's foreign minister has denounced as a "dangerous escalation" the White House allegations that Syria is preparing a new chemical weapons attack.
Defence Secretary Jim Mattis insisted the United States was not taking sides in the Syrian conflict.
"When they don't need certain things any more, we'll replace those with something they do need", said Mattis, who is expected to meet with his Turkish defense counterpart during a North Atlantic Treaty Organisation meeting later this week in Brussels. "We try to end that through diplomatic means". The series of air attacks, taken in self-defense by USA forces, according to the Pentagon, occurred as pro-Assad forces threatened or approached US -backed Syrian fighters.