The spillage was caused by oil that had got into a drain and affected a nearby pond close to Potteric Carr nature reserve.
A spokesman for the Environment Agency said initial investigations suggested that oil had entered the watercourse from a drainage system linked to an industrial estate.
They added: “The Environment Agency is investigating a pollution incident in a watercourse at White Rose Way, Doncaster, in which oil has entered a drain and affected a nearby pond.
“Our officers attended the site over the weekend to assess the impact on the environment and to investigate potential sources.
“It appears that the oil has entered the watercourse from a drainage system linked to an industrial estate.
“We used booms and pads to clean up the oil and to prevent it from spreading further downstream, and we have taken samples for laboratory analysis.
“Our officers have also prevented the oil from entering Potteric Carr Nature Reserve by disabling the pumps that feed it water from the affected pond.
“We are continuing our investigations into the incident this week, using dye testing to check drain connectivity to the watercourse.”
“While the dye testing is taking place, people should not be concerned if any watercourses in the White Rose Way area appear to be bright green.
“The dye we use is a harmless substance that is commonly used in food and medical applications.”
Following the discovery of the spillage on Friday, officers from the Environment Agency worked throughout the weekend to tackle the diesel spillage.
Commenting on Twitter, Paul Reeves of the Environment Agency said of the operation: “We have had teams working near Doncaster today dealing with a major oil pollution. We have contained and removed a significant amount of diesel.”