It happened on Thursday afternoon in the Grange River, a tributary of the Moyola.
Members of the local angling club said a significant stretch of the river near Desertmartin had been affected.
The NI Environment Agency said it was alerted by a member of the public at 4 pm on Thursday that there was slurry and dead fish in the water.
“An NIEA Inspector is currently working with the site owner to try to prevent further run off from the premises,” a spokesperson said.
“NIEA have collected statutory samples and the joint investigation with DAERA Inland Fisheries is ongoing.”
The Moyola is a 27km-long river that cuts through the heart of mid-Ulster, rising in the Sperrins and flowing into Lough Neagh.
An attempt will be made later to count the dead fish and assess the environmental impact of the incident.