The three men, who are all aged in their 50s, were injured in an incident in Woodside Road, Glenrothes at about 1am today.
One of the victims, aged 50, is in a critical condition in the Western General Hospital in Edinburgh, Police Scotland said.
The other two men were taken to the Victoria Hospital in Fife for emergency treatment.
Police are appealing for any witnesses to the disturbance to come forward.
Detective inspector Colin Robson is leading the investigation and said: "Our early inquiries have revealed that there was an disturbance in the area of Woodside Road, Glenrothes near to Dovecot Park, in the early hours of the morning.
"We're carrying out door-to-door inquiries around the area to try to find out more information from local residents but I'd like to further this by appealing directly to the wider public for their assistance."
He added: "We have extra officers dedicated to investigating the incident and have increased high visibility patrols in the area."