Kerosene leaked from a pipeline near Nantycaws, Carmarthenshire, into nearby Nant Pibwr, with dead fish spotted.
The Welsh Government said the A48 will close in both directions from the Nantycaws turning to Carmarthen from 19:00 BST on Friday until 06:00 Monday.
Traffic will be diverted through Nantycaws and Llangunnor.
Rural Affairs Secretary Lesley Griffiths visited the site of the spill on Saturday.
In a letter to assembly members, she confirmed the scale of the spill was bigger than previously thought.
Details of when a main road will close for repair work to a damaged fuel pipeline that leaked more than 140,000 litres of oil have been confirmed.