Trump, on social media, accused the industry of contributing to the high cost of beef for US consumers "through Illicit Collusion, Price Fixing, and Price Manipulation".
His call for a probe focused on potential antitrust violations comes as soaring beef prices have become a political problem, threatening to undercut his promises to bring down food costs in the US.
The announcement also follows Republican losses in key elections this week, where voters' concerns about the cost of living and Trump's handling of the economy boosted Democrats.
Trump has put beef prices at the centre of his messaging about the cost of living - and food inflation, specifically. But his other recent proposals to lower prices sparked backlash among ranchers.
Last month, on social media, he urged US ranchers to lower prices for their cattle. Then, he drew their ire when he proposed importing more beef from Argentina, potentially quadrupling US purchases.
At the time, ranchers worried that some of his solutions would make it harder for them to make a living, while not helping much at the grocery store.
As Trump keeps pushing to lower beef prices for American consumers, his latest call for federal investigation signals a possible shift in focus, toward the meat-packing industry, which slaughters livestock and then processes and packages their meat.
"We will always protect our American Ranchers, and they are being blamed for what is being done by Majority Foreign Owned Meat Packers, who artificially inflate prices, and jeopardize the security of our Nation's food supply," Trump wrote in a social media post.
Retail prices for beef mince rose 12.9% in the 12 months to September, and beef steaks were up 16.6%, according to government data published in October.
A pound of ground chuck - richer mince from the neck and shoulder of cows - now costs an average of $6.33 (£4.75), compared to $5.58 a year ago.
The increases significantly outpaced general food inflation, which stood at 3.1%.
Some economists point to supply contraints, combined with robust demand for beef, as the main reason for high prices.
The country's cattle inventory has fallen to its lowest level in nearly 75 years. Decades of contraction in the industry has worsened as several years of drought have forced ranchers to slash their herd sizes - and herds take several years to rebuild.
But others argue the US government could lower beef prices if it focused on the way a handful of companies dominate the market for meat processing.
Today, just four firms control more than 80% of the beef slaughtering and packing market. The meat processing firms - Tyson, JBS, Cargill and National Beef - have faced several lawsuits, including one filed by McDonald's alleging that they colluded to inflate the price of beef.
Consolidation in the meat processing industry has elicited bipartisan scrutiny.
The Biden administration issued an order directing agencies to tackle corporate consolidation across the food supply chain, though Trump revoked that order earlier this year.
Former President Joe Biden also asked the Agriculture Department to investigate big meat packers, accusing the firms of of raising prices and boosting their profits during the pandemic.
