Cleaners on First Great Western trains at the main stations in Cardiff and Swansea have gone on strike for 24 hours in a dispute over pay and pensions.
The RMT union said the workers, who are employed by Mitie, are low-paid and badly treated.
Mitie said it believes it has settled all outstanding issues with the union.
The rail firm said it will not disrupt its south Wales to London services.
The RMT said the industrial action was in response to its dispute with the firm over "wage discrepancies" suffered by its members.
It said its Mitie members had delivered a unanimous vote in favour of striking.
RMT General Secretary Bob Crow said: "This appalling situation shines the spotlight on just how these facilities companies operate and how they treat their low-paid staff.
"The company has seen the unanimous vote for strike action, which reflects the anger amongst their staff, and it's now up to Mitie to step up with a line-by-line resolution to each of the issues we have raised in this dispute."
A spokesman for Mitie said: "All issues between Mitie and the RMT referred to on the original ballot have been resolved."
"Mitie has not been formally advised by the union of any further issues which would warrant a strike action this coming Friday and is seeking clarification."
FGW said it did not anticipate delays to its services on Friday as a result of the dispute between the RMT and Mitie.
A spokesperson said: "It won't affect any of our services. Mitie will be providing its service as normal.
"There was a previous strike on 4 February and there was no disruption to the service on our trains. Our customers did not experience any disruption."
RMT train drivers for Arriva Trains Wales, which operates in the south Wales valleys, had planned to strike on 4 February, the day of the Six Nations rugby clash between Wales and England at the Millennium Stadium in Cardiff.
That action was called off on legal advice to the union after the rail firm launched legal proceedings.