Foreign Minister Penny Wong said staff were directed to leave "due to the deteriorating security situation".
"Essential Australian officials will remain in-country to support Australians who need it," she said in a statement posted on social media on Thursday night.
"We continue to advise Australians not to travel to Israel and the UAE.
"We urge you to leave the Middle East if you can and if it's safe to do so."
Senator Wong's statement comes after the government told families of Australian officials in Israel and Lebanon to leave in the days before the war began on February 28.
Voluntary departures were also offered to diplomats' dependents in the UAE, Jordan and Qatar.
The war has so far killed about 2000 people and caused the biggest disruption to global oil supplies in decades.
Oil prices that had come down earlier this week after US President Donald Trump said the war would soon be over soared back above $US100 a barrel after an apparent escalation in Iranian attacks on shipping in the Persian Gulf.
The two-week-old conflict has prompted thousands of Australians to flee the region, with the government saying on Tuesday that more than 2600 Australians were returning home on commercial flights.
Senator Wong said the overwhelming majority of Australian citizens who were travelling through the Middle East and became stuck when the war broke out had returned home.
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