Lomond Court, in the Woodside area of Glenrothes, is said to have found the cases during a single round of testing, with many staff now self-isolating after the outbreak.
It is understood that there have been less than five deaths at the Lomond Court home related to the coronavirus outbreak.
HC One, the company which runs the care home, said that it was working with NHS Fife in order to respond to the outbreak.
A spokesperson for the home paid tribute to those affected, saying: “Our thoughts and sympathies are with all families who have lost a loved one from coronavirus and we are doing our utmost to support them during this difficult time.
“We are working closely with our local health partners including NHS Fife to respond to the outbreak.
"We continue to implement our infection control measures and we have the staff, PPE and equipment needed to protect residents and colleagues.
“Our Residents are our absolute priority and we are doing everything we can to support them to return to good health.”
In a statement, NHS said: “NHS Fife can confirm that 26 residents and 22 staff associated with Lomond Court Care Home in Glenrothes have tested positive for COVID-19.
“Sadly, there has been loss of life connected to the cluster.
"For reasons of confidentiality we are unable to release any further information on this, however, we can confirm that the number is less than five. Our thoughts are with the bereaved at this time.
“Lomond Court Care Home is currently closed to visitors and new admissions.
“NHS Fife’s Health Protection Team and the Fife Health and Social Care Partnership are working alongside Fife Council’s Environmental Health Service to support to the management of the care home and help reduce further transmission of the virus.”
Fife is currently under level 3 restrictions, with 79 new cases recorded overnight on Friday.
Across the Kingdom there are currently 36 people in hospital who have tested positive for coronavirus.
A total of 4394 cases have now been detected in Fife since the start of the covid-19 outbreak.