The Transport Salaried Staffs Association accused First Great Western of "daylight robbery" after claiming that the firm planned to raise fares from September 8 for advance tickets bought at stations rather than online.
The company, which runs services between London and the South West and South Wales, denied the claim, saying there was no single set price for an advance purchase ticket because prices varied depending on how busy a train was.
Manuel Cortes, the union's general secretary, said: "This is yet another move to close ticket offices by driving passengers online when trying to find cheaper fares.
"They are penalising all passengers who buy advance tickets at a station and that is particularly unfair on the middle aged and elderly who prefer to use their local booking office."
The union is writing to First Group chief executive Tim O'Toole asking if rises will also apply at the company's four other franchises - First Capital Connect, TransPennine Express, First Hull Direct and ScotRail.
"These fares already offer significant discounts of more than 60% on standard walk up fares, and this will not change in September.
"Like many other operators, we are currently running a promotion that gives customers an additional 10% discount on these fares when booking from our own website."