Staff are protesting against short-term contracts and limited career progression, along with a lacklustre pay deal with a below-inflation increase.
The strike has forced ABC's flagship news programs including Radio National Breakfast off air, instead playing pre-programmed shows and BBC content.
Journalists have been advised not to speak to the media ahead of the strike, which begins at 11am AEDT on Wednesday.
Large gatherings are expected outside ABC offices nationwide, including in Melbourne and Sydney, during the 24-hour strike.
The Community and Public Sector Union and the Media, Entertainment and Arts Alliance are representing staff taking action at the ABC.
"We've been in bargaining for a long time now with the ABC and what we want to see is a pay off that reflects cost-of-living pressures and actually respects the really important work that the ABC do," CPSU national secretary Melissa Donnelly told AAP.
"ABC plays such an important role in our society and in Australian storytelling and it's really important ABC management come to the table."
About 60 per cent of ABC staff rejected management's offer that included a 10 per cent pay rise across three years.
The unions are also demanding greater night-shift penalty rates, reproductive health leave, and rules relating to artificial intelligence.
"Experienced journalists and media workers are being asked to do more with less – with fewer opportunities for pay progression, less certainty about their future, and growing workloads," MEAA chief executive Erin Madeley said.
It will be the organisation's first major strike since 2006.
ABC managing director Hugh Marks said the deal was financially responsible and competitive for the industry.
"The average tenure of an ABC staff member is more than 10 years, which is three times the economy average ... over 90 per cent of ABC staff are ongoing employees," Mr Marks said.
"The pay offer reflects the maximum level the ABC can sustainably provide and is balanced when looking across all the factors that we need to consider."
More than 4400 people work at the ABC, including 2000 in news, the largest division.
The ABC has been contacted for comment.
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