Anthrax has been confirmed by veterinary officials on a cattle farm north-east of Shepparton.
Agriculture Victoria said the affected farm was a single property which was now confined under a quarantine order.
Following veterinary investigation, the infectious zoonotic bacterial disease was determined as the cause of death in five cattle on the farm.
The carcases of the dead livestock were safely disposed of and the remaining cattle on the property have been vaccinated.
Neighbouring farms have also been informed.
Victoria's chief veterinary officer Dr Graeme Cooke said due to early reporting by the impacted farmer and veterinarians, swift action was taken to reduce the likelihood of the disease spreading to more livestock.
"Incidents of anthrax detected in cattle and sheep in the region occur during the warmer months when it is drier and cattle and sheep forage deeper into the soil when grazing," he said.
"Quarantine controls will not affect the movement of local people, vehicles, or livestock."
Agriculture Victoria said its investigations remained ongoing.
"This incident again highlights the importance of early reporting and the need to consider anthrax as a differential diagnosis whenever unexplained sudden death occurs in sheep, cattle or other susceptible species in Victoria," an Agriculture Victoria spokesperson said.
Anthrax is an infectious zoonotic bacterial disease of animals, caused by the spore-forming bacterium Bacillus anthracis.
Although the disease usually appears during the warmer months, cases of anthrax may occur at any time of year and in locations where the disease may not have been recognised for many years.
Cattle and sheep were infected through ingesting spores present in contaminated soils.
Once ingested, the bacteria produce potent toxins that cause the clinical signs of anthrax including fever, inappetence, lethargy or sudden death.
Farmers who suspect Anthrax on their farms were reminded that it is a notifiable disease.
Any suspected case should be reported immediately to local Agriculture Victoria staff or via the all-hours emergency animal disease watch hotline on 1800 675 888.