NSW Health has confirmed a man in his 70s from Griffith who died last month had the Japanese encephalitis virus (JEV).
The man died on February 13, and after post-mortem testing, it was confirmed he had the disease, which is spread by mosquitoes.
The man was confirmed as the third known case of JEV in NSW, and more people were undergoing testing.
The virus was first detected in the state last month, and authorities were urging people to protect themselves against mosquito bites.
JEV had never been previously detected in humans in NSW.
Two other cases, a man and a child from different towns in the state's south, were confirmed on Monday.
The virus is potentially fatal for humans but symptoms develop in only 1 per cent of cases.
It has now been detected across four states, in what are the first known cases on the Australian mainland.
The region around the NSW-Victoria border has been identified as an area of concern for health authorities.
Earlier this week a man in his 60s died from JEV in Victoria.
The outbreak has been declared an incident of national significance by the Acting Chief Medical Officer, Sonya Bennett.
Dr Bennett said JEV was typically considered a travel disease but there had been past cases in the Torres Strait Islands.
"This outbreak we have known about for just over a week, and what is concerning is that we became aware of a large number of infected piggeries at the same time," she said.
Dr Bennet said authorities would be in touch with people in affected areas, and vaccines were being rolled out for piggery workers.
"We had a lot of flooding and people should certainly be protecting themselves against mosquitoes by wearing long, loose-fitting clothing, using repellent and applying when necessary," she said.
