More than 150 porters, cleaners, and catering staff at Carlisle's Cumberland Infirmary have taken strike action over payments for working unsocial hours.
NHS workers receive enhanced rates for working at unsociable times such as weekends or through the night.
Unison says non-medical staff at the hospital have not been getting these payments for 10 years.
Mitie, which took over the contract for those workers in November, said talks were "continuing".
The workers, all members of Unison and the GMB union, plan further action on Monday.
Unison said it followed two days of "crunch talks" mediated by the Advisory, Conciliation and Arbitration Service (Acas).
Both trade unions offered to withdraw the action if they received a written guarantee from Mitie that workers would begin to receive unsocial hours payments within the next two months, but this was not forthcoming, it said.
David Atkinson, from Unison, added: "It is incredibly disappointing that key workers, who have put their lives at risk to keep us safe during this pandemic, have been forced to take strike action by their intransigent employer Mitie.
"Having risked their lives for the last year, this committed group of hospital cleaners, caterers, porters and switchboard staff are determined to secure the unsocial hours payments they are rightly owed."
A Mitie spokeswoman said the firm had only been "privy to this contract for a few months" having acquired previous holder Interserve at the end of November 2020.
"As Unison is well aware, we are not in a position to provide a guarantee at this time," she said.
She added there were "continuing talks to resolve the situation and agree a long-term solution for all parties".
North Cumbria Integrated Care NHS Foundation Trust said in a statement it was "encouraging" the union to work with Mitie to resolve the dispute.
It added: "We are working closely with Mitie's local team to ensure arrangements are in place to sustain safe services despite the action taking place."