A biosecurity team at the federal agriculture department detected the fire ant nest at the Enfield Intermodal Centre in Sydney's inner west suburbs.
The nest was quickly treated at the logistics hub, a facility for the freight industry, to prevent an infestation from taking hold.
Genetic analysis undertaken by the National Fire Ant Eradication Program confirmed the species as red imported fire ants (Solenopsis invicta) and are likely to have originated from overseas via a container.
NSW Agriculture Minister Tara Moriarty said properties within a 500m radius of the freight facility would be inspected to keep to assure the community fire ants were being eradicated.
"It's important to note that no further ants have been found, but we are focused on making sure any potential spread is identified and eradicated quickly," she said on Tuesday.
"While fire ants are one of the world's most invasive species, they can and have been successfully eradicated in NSW."
The Invasive Species Council warned the spread of the "super pest" could cost the Australian economy up to $2 billion a year.
The federal department's entomologists confirmed the red and brown ants - two to six millimetres in length - are not related to the only other fire ant-impacted site in the country in southeast Queensland.
Further surveillance and treatment will be expanded to a 2km radius of Sydney suburbs in the coming weeks.
Fire ants were detected at Port Botany in 2014 and were successfully eradicated.
The council's advocacy director Reece Pianta warned the risk of fire ants spreading beyond Queensland's borders was still alive.
''This detection is a reminder of why we need to keep investing in strong border biosecurity and surveillance," he said.
"Fire ants are widespread among many of Australia's trading partners, so finding new incursions quickly is critical to stopping them becoming established."
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