The management of Flicks restaurant at Cityside Mall on York Street have voluntarily closed the premises and are cooperating with the investigation.
Eleven confirmed or suspected cases have been identified.
Anyone who ate at the restaurant since 24 September and has had symptoms of diarrhoea and/or abdominal pain should contact their GP urgently.
Environmental health officers from Belfast City Council are also taking part in the investigation.
The PHA said symptoms of E. coli O157 can include diarrhoea, which can range from mild to profuse, watery or bloody diarrhoea, stomach cramps, nausea or vomiting.
The agency's Dr Michael Devine said, on average, symptoms last for about two weeks.
He said the first case was discovered on 9 October and the restaurant closed on 11 October.
"We alerted general practitioners on Friday that there had been an increase in E. Coli in the Belfast area and then subsequently, on Saturday, we advised particularly of the association with Flicks restaurant," he said.
"At this stage it seems to be mainly adults involved, but obviously our investigations are ongoing and it may be that children have been affected.
"At this point, what we're trying to do is encourage people if they have eaten at Flicks restaurant, particularly on or around 4 October, and they've got symptoms of diarrhoea, particularly bloody diarrhoea, or tummy pains, that they would get medical advice from their GP."
Dr Devine said four of the 11 people known to have been infected had been in hospital, but he understood all had since been discharged.
He said some people could have been infected but not be experiencing any symptoms.
"With E. Coli 0157, it can be a very serious infection and that's why we're raising this alert, but some people can have very minimal or even no symptoms," he said.
"So if people have eaten at the restaurant and they've got concerns, I would advise they get medical advice."
In a statement, Flicks restaurant said: "We are fully co-operating with the Public Health Agency's investigation into a recent E. coli outbreak.
"As soon as they contacted us regarding the investigation we immediately closed the restaurant.
"We will continue to assist the PHA in finding the source of this outbreak."
