Drivers, guards and staff at First Great Western (FGW) are to be balloted on strike action over use of agency staff and unfair dismissal claims.
Railway Maritime and Transport (RMT) members will vote on a campaign of industrial action in the coming weeks.
The announcement comes on the day FGW cleaners walked out for 24 hours over pay and zero-hour contracts.
The company, which has just been given an extension to its contract, said it was working to meet its obligations.
However, the RMT claims there has been a breakdown of industrial relations at the firm and around 2,500 of its members will be balloted over issues including claims of a high number of unfair dismissals.
'Have money available'
RMT general secretary Bob Crow said its members had "had enough of being kicked from pillar to post".
"With First securing a highly lucrative extension to their Great Western contract last week there is no excuse for them to blame the failure to reach agreement on the uncertainty over the franchise," he said.
"It is also clear as day that they have the money available now to employ adequate numbers of permanent staff and to treat them with dignity and respect in every workplace."
Earlier, cleaning staff from the RMT union and contractor MITIE began a 24-hour walk-out.
They claim to be the operator's lowest-paid workers and earlier this year, rejected an offer of a 2% wage rise.
The strike is due to run until 05:59 on Saturday.
An FGW spokesman said the firm was working with MITIE, as its contractor, to ensure it met its contractual obligations.
The firm operates trains across the Western region, carrying 1.5m passengers every week on 9,000 services and calling at 276 stations.