The spillage - believed to be diesel - has affected about 3.5miles (5.5km) of the River Kennet.
The source of the pollution is thought to have originated in the industrial areas in south Reading, the Environment Agency said.
Oil absorbent booms have been deployed at Foudry Brook near Grazeley to try and help remedy the situation, a spokesman added.
Wendy Herman, from animal charity Swan Support, said 50 swans had been rescued but a further 10 were still outstanding.
"It's a massive area with diesel all over the place.  It's really bad - and it's not just how [it will affect the way] the swans look, it's what they're ingesting, so it's really important we get as many out of the water as possible."
The agency said it was investigating the incident with the help of police.
About 50 swans had to be rescued after oil leaked into a river in Reading.