Some residents will have to travel further to do their banking in person.
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ANZ Mittagong, located outside of Highlands Marketplace shopping centre, was closed temporarily in April.
The company informed staff the bank wouldn't reopen its doors several weeks ago.
An ANZ spokeswoman said the company "is seeking to retain as many staff members as possible".
"[This is] through helping them move into a range of new roles across ANZ where possible, either supporting customers in other ways or in nearby branches," the spokeswoman said.
The spokeswoman said the closure of the branch was due to the popularity of online and mobile banking.
"Each year we see a 10 per cent reduction in transactions," she said.
"At the same time demand in many of our non-branch roles such as customer contact centres and the operations team which manages hardship and other enquiries has grown dramatically."
The closure has drawn a mixed response from residents on social media.
Some customers raised questions about where they could deposit their cash without a fee, while others were concerned about the distance they had to travel to their second closest branch.
"We recognise the transition will be more challenging for some customers," the ANZ spokeswoman said.
"We have written to customers personally to let them know how they can use alternative banking methods, including online, telephone and mobile banking."
ANZ closed its Moss Vale branch in March 2016.
The Bowral branch is the last ANZ bank in the Southern Highlands.
