British Transport Police are seeking the pair who carried out the attack between Mexborough and Conisbrough stations on September 19.
The conductor was checking tickets on the 10.24am Sheffield to Scunthorpe train when he saw a man and a woman head for the nearby toilets.
At Conisbrough, he opened the toilet door and asked them to leave as they didn’t have any money or tickets.
A woman went over to the conductor and asked if he needed any help. The couple then both spat at her and shouted abuse. The pair then left the train at Conisbrough.
If you know the couple in the image, contact 0800 40 50 40 or text 61016, quoting reference 1700065642.