A second woman has died from Murray Valley encephalitis and a man is in hospital with the virus as more suspected cases are investigated.
A woman in her sixties died in late February and a man in his seventies is receiving treatment in hospital, Victoria's health department says.
It follows the death of a woman in her 60s who lived in the Buloke Shire in the state's northwest in early February.
A total of three cases of the virus and two deaths have been reported in Victoria this mosquito season.
A number of suspected cases are under investigation, the health department said in a statement on Monday night.
Both the confirmed new cases were likely exposed to mosquitoes in northern Victoria
There is no vaccine against Murray Valley encephalitis but the virus is known to be circulating in mosquito populations in northern Victoria.
It can cause a rare but potentially serious infection of the central nervous system, and people in northern Victoria are being urged to reduce their risk of mosquito bites.
