Cleaners at Southampton General Hospital have begun a two-day walkout.
About 240 cleaners are striking in a dispute with Southampton University Hospitals Trust over backdated pay.
The dispute dates back to a 2006 agreement to pay contract staff the same wages as their NHS-employed colleagues, who were earning more.
The agreement comes into force in April 2011 but the the cleaners and unions say the money should already have been passed on.
The hospital trust said it was not obliged to increase the wages and could not afford to until next year.
Steve McManus, chief operating officer for Southampton University Hospitals NHS Trust, said: "We very much want to make sure that our cleaning staff get a fair wage for the job they do.
"We don't get a ring-fenced sum of money for this and we have to make difficult choices and in this situation we've been absolutely committed to make sure that our cleaning staff, who we really value, do get a fair wage but we have had to do that over a period of time."
The trust said high-risk clinical areas would continue to be cleaned throughout the industrial action.