The latest challenge was filed with the Federal Court on Wednesday.
"eSafety is aware X has filed a judicial review in the Federal Court as to whether eSafety's Relevant Electronic Services (RES) standard should apply to its X platform," a spokesperson for the regulator said on Thursday.
"The RES standard requires online platforms to take meaningful action to tackle the worst of the worst online content, including child exploitation and abuse material.
"eSafety is prioritising its efforts on ensuring all platforms and services meet their obligations under the Online Safety Act to address high-impact and harmful content to keep Australians safe online."
It is not the first time controversial billionaire Elon Musk, who owns X Corp, has been involved in a legal tit-for-tat with Australian authorities.
The online safety regulator issued a financial penalty of $610,500 to X in February 2023, alleging the social network failed to adequately respond to questions about how it tackled harmful content on its platform, including child sexual abuse material.
X Corp appealed, in ongoing court proceedings, arguing it did not exist when the commissioner issued a transparency notice to Twitter regarding child sexual abuse material.
In April 2024, the commissioner issued an edict to X, formerly known as Twitter, to remove graphic content after clips of Sydney Bishop Mar Mari Emmanuel being stabbed remained on the platform.
During the months-long saga, Mr Musk accused the Australian government of suppressing free speech.
He called the Australian government "fascists" over attempts to tackle deliberate lies spread on social media.
Several politicians hit back, with Prime Minister Anthony Albanese labelling Mr Musk an "arrogant billionaire".
The safety commissioner later discontinued the Federal Court proceedings.
The agency said it was assessing the latest court documents filed by X Corp and would consider its next steps.
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