Ohio-based Wendy's, the third-biggest fast-food burger chain in the world, unleashed the humorous campaign across the US to promote its use of "100 per cent North American" beef patties.
Rivals including McDonald's import Australian beef to the US.
The Wendy's TV commercial, which has been receiving big air time in the US, includes three tough Aussie rugby players, with one gruffly asking: "What's wrong with Australia?"
The ad then points to how Wendy's uses fresh, never frozen 100 per cent North American beef while some of its competitors import their beef from Australia.
In another playful dig at Australia, the ad ends with: "Score one for the guys without cool accents."
Meat & Livestock Australia's North American business manager David Pietsch described the Wendy's campaign as "a light-hearted ad to push" Wendy's position.
He said Australian meat producers had many loyal American customers because of Australia's longstanding reputation for sending fresh and frozen quality beef to the US.
"There's a number of companies in the US that use a proportion of Australian beef that is very lean to mix in with their fattier domestically produced beef to make the perfect formulation for a hamburger," Mr Pietsch told AAP.
The Wendy's campaign, via the website www.othr-guyz.com, also pokes fun at rival chains' use of frozen burger patties with the faux product Freezy Diskz!
The Freezy Diskz! is described as a multi-function beef product that is so hard and cold it can also be used as: a hockey puck; a door stopper; to level a table; by a judge in court to slam a gavel on; a drink coaster; and to hit at karate practice.
The ad also includes former Incredible Hulk actor and bodybuilder Lou Ferrigno, who shows how he can throw a frozen beef patty to stop a "bad guy".