"Overnight the Australian Air Task Group operating in the Middle East attacked its first target in Iraq," the ministry said in a statement.
"Two bombs were dropped from an F/A-18F Super Hornet on to an ISIL facility. All aircraft exited the target area safely and returned to base."
Australia is part of the international coalition conducting an air campaign against the IS group that the United States has been building since first launching air strikes in August.
Jets flew their first armed mission on Sunday, but no bombs were dropped until now.
The defence ministry said no further details of the mission were available.
Australia has also sent 600 troops to the UAE, including special forces, primarily to help Iraqi forces with planning and coordination against IS. They are awaiting final legal approval before deploying.