Federal prosecutors said brothers Eric and Ryan Jensen were arrested on misdemeanour charges of introducing adulterated food into interstate commerce. Each man faces six counts.
They pleaded not guilty in federal court and were released on unsecured bonds. Trial is scheduled for Dec. 2.
Prosecutors said the federal Food and Drug Administration and the Centers for Disease Control and Prevention determined the Jensens didn't adequately clean the cantaloupe.
Criminal charges in food poisoning cases are rare, said attorney William Marler, who represents many of the listeria victims in civil cases against Jensen Farms. Only four other people have faced such charges in the past decade, he said.
The FDA has said the melons likely were contaminated in Jensen Farms' packing house. It concluded that dirty water on a floor, and old, hard-to-clean equipment probably were to blame.
The epidemic was the deadliest outbreak of foodborne illness in 25 years, and it delivered a serious blow to Colorado cantaloupe farmers.
A number of lawsuits were filed by people who were sickened or who had a family member die after the outbreak.
Eric Jensen, 37, and Ryan Jensen, 33, could face up to six years in prison and up to $1.5 million in fines each if convicted of all counts against them, prosecutors said.
The Jensens' farm in southeastern Colorado filed for bankruptcy after the outbreak.
After Thursday's hearing, the men released a statement calling the outbreak a "terrible accident" and saying they were shocked and saddened by it. The statement said the charges do not imply they knew about the contamination, or that they should have known about it.
Jeff Dorschner, a spokesman for federal prosecutors, said the misdemeanour "was the best, most serious charge we could find."
Felony charges would have required prosecutors to show the contamination was intentional.
"The real significance of the case against the Jensens is they are being charged with misdemeanours, which do not require intent, just the fact that they shipped contaminated food using interstate commerce," Marler said.
Dorschner said prosecutors decided to pursue the case because so many people were affected.
"It was the magnitude of the number of people who were hospitalized and the number of people who died," he said. Prosecutors said people in 28 states ate the cantaloupe, and 147 people were hospitalized.
The illnesses quickly were linked to the Jensens' farm, with the FDA saying on Oct. 19, 2011, that the outbreak probably was caused by pools of water on the floor and old, hard-to-clean packing equipment there. The agency said contamination at the packing facility likely was to blame. Investigators found positive listeria samples on equipment and fruit there.
The FDA said Jensen Farms had recently purchased used equipment that was corroded and hard to clean. The agency said the way the cantaloupes were cooled after coming off the fields might also have contributed to listeria growth.
Asked why it took so long to file charges, Dorschner said officials needed time to develop the case.
The outbreak was a setback for farms in Colorado's revered Rocky Ford cantaloupe region, where hot, sunny days and cold nights produce fruit known for its distinct sweetness.
Jensen Farms was about 90 miles away from Rocky Ford, but the Jensens used the Rocky Ford name, and sales dropped across the region. Later, Rocky Ford farmers registered Rocky Ford Cantaloupe as a trademark, hired a full-time food safety manager and built a central packing operation where melons are washed and rinsed.
Tammie Palmer, whose husband, Charles, became ill after eating the cantaloupe, said she hopes the Jensens never return to farming.
The Palmers, represented by Marler, filed a lawsuit against Jensen Farms seeking $2 million. The suit was still pending when Charles Palmer died this year of cancer.
"I was hoping everything would be settled and I could do something with my husband, but that's not going to happen," she said.
Associated Press Writer Kristen Wyatt contributed to this report.