Medecins Sans Frontieres says hospitals it supports in Syria treated about 3,600 patients with "neurotoxic symptoms", of whom 355 have died.
The medical charity said the patients had arrived in three hospitals in the Damascus area on 21 August - when opposition activists say chemical attacks were launched against rebels.
But MSF says it cannot "scientifically confirm" the use of chemical weapons.
Both sides in the conflict accuse each other of using them.
MSF says staff at the hospitals described a large number of patients arriving in the space of less than three hours with symptoms including convulsions, dilated pupils and breathing problems.
It said many were treated with atropine, a drug administered to those with "neurotoxic symptoms".
"MSF can neither scientifically confirm the cause of these symptoms nor establish who is responsible for the attack," said MSF Director of Operations Bart Janssens.
But it added that the symptoms, as well as the "massive influx of patients in a short period of time" strongly suggest mass exposure to a neurotoxic agent.
"This would constitute a violation of international humanitarian law, which absolutely prohibits the use of chemical and biological weapons," MSF said.
The Syrian Observatory for Human Rights - a British-based group that monitors the conflict - estimated that 322 had died, 54 of them children.
In the immediate aftermath, casualty figures varied widely with opposition activists saying between several hundred and more than 1,000 had been killed.
Correspondents say MSF's statement adds to mounting allegations that chemical weapons were used in suburbs to the east of Damascus and in an area to the south-west on 21 August.
Unverified video footage posted soon afterwards shows civilians, many of them children, dead or suffering from what appear to be horrific symptoms consistent with a chemical attack.
Rebels and opposition activists accuse forces supporting President Bashar al-Assad of carrying out such attacks.
But state TV accuses the rebels, saying barrels of chemical weapons were found as troops entered previously rebel-held districts.
The international community is split on the issue. Russia and Iran, both allies of Damascus, have separately accused the rebels of using chemical weapons.
But France's Foreign Minister Laurent Fabius said on Saturday that all available information indicates that the government of Bashar al-Assad was responsible.
UK Foreign Secretary William Hague has also said he believes this was "a chemical attack by the Assad regime".
In phone call on Saturday, US President Obama and UK Prime Minister David Cameron reaffirmed their "grave concern about the reported use of chemical weapons by the Syrian regime against civilians near Damascus on Wednesday", the White House said.
The two leaders agreed to continue consulting closely on the incident and on possible responses by the international community to the use of such weapons, it added.
The UN disarmament chief Angela Kane has meanwhile arrived in Damascus to press the Syrian government to allow access to the sites of the alleged attacks.
A team of UN weapons inspectors has been in Syria since 18 August to inspect three sites, but Damascus has not yet said whether it will allow them to expand their visit.
The UN says more than 100,000 people have been killed since the uprising against President Assad began more than two years ago.
- 01:15: 21 August (10:15 GMT 20 Aug): Facebook pages of Syrian opposition report heavy fighting in rebel-held districts of Ghouta, the agricultural belt in eastern Damascus
- 02:45: Opposition posts Facebook report of "chemical shelling" in Ein Tarma area of Ghouta
- 02:47: Second opposition report says chemical weapons used in Zamalka area of Ghouta
- Unverified video footage shows people being treated on pavements in the dark and in a makeshift hospital
- Reports say chemical weapons were used in Ghouta towns of Irbin, Jobar, Zamalka and Ein Tarma as well as in Muadhamiya to the west, but this is not confirmed
- Syrian government acknowledges military offensive in the Ghouta area but denies chemical weapons use