A charity which looks after the River Irwell says a 25-mile stretch has been left devastated after a second pollution incident in three weeks.
Volunteers from the Mersey Basin Rivers Trust found white foam in the water at Burrs Country Park in Bury on Sunday.
A suspected pesticide dump earlier this month killed fish and insects between Manchester and Rawtenstall, Lancashire.
The Environment Agency said it was investigating and did not yet know the cause of the incidents.
Trust spokesman Mike Dudd said the loss of insects was important to the river's ecosystem.
"It's going to take the river a long, long time to recover and in the meantime there's going to be a lot of fish, bats and birds go hungry.
"The recovery of the River Irwell has been one of the biggest environmental recovery stories of the last 25 years - the river polluted so badly by the Industrial Revolution now holds the country's best stocks of brown trout and other fish which support kingfishers, otters and dippers.
"This latest event is a massive setback for the river and all those who work so hard to bring these improvements about."
The Environment Agency confirmed samples of water had been taken for analysis.
A spokesman said: "The data from these samples has enabled the team to narrow down the search area for the source of the pollution.
"At this time we have not received any reports of fish mortalities and are treating the two incidents separately."