Sarah Langhorne died in 1871, but many people claim to have seen her ghostly apparition peering from a window of Altona Homestead.
The homestead, built by Sarah and husband Alfred in 1842, has long had a reputation for being haunted and has attracted international attention from paranormal investigators.
Now Lantern Ghost Tours will run guided tours of the homestead from next month as part of the National Trust Heritage Festival.
Company founder Jacqueline Travaglia says there have been numerous sightings of a forlorn Mrs Langhorne, who missed city life and whose beauty stopped her from making friends.
Her life was marred by tragedy after she lost two of her children.
“Sarah gave birth to Henry Langhorne at the homestead,” Ms Travaglia said. “He died aged seven months and she also lost a teenage daughter.
“Although Sarah is long departed, people have seen a sad woman’s face staring out from the lounge room window.”