The incident, in March 2017, killed a large number of fish, invertebrates, and eels in the Tuffley Brook, Gloucester.
It was caused by blue tissue from the baby changing area on Gloucester Gateway’s premises being flushed down toilets and causing a blockage, instead of being disposed of in bins.
The company, which owns Gloucester M5 Services, no longer provides the tissue in these toilets.
The Trust will use the funds to pay for a sustainable drainage system (SuDS), designed to show that SuDS can be attractive and functional within a landscape and emphasise the importance of careful water management.
Ian Skuse, officer at the Environment Agency, said: “Enforcement undertakings are an effective enforcement tool used by the Environment Agency to enable companies to address breaches of environmental legislation while supporting suitable environmental projects.
“The contribution to Gloucestershire Wildlife Trust will help enhance the site, while providing an example of how modern drainage systems can work with the natural water cycle.”