Investigators initially said those accused of wrongdoing in the 2013 collapse of the Rana Plaza building would be charged with culpable homicide, which carries a maximum punishment of seven years in jail.
However, they later changed the charges to murder because of the gravity of the disaster.
Masud Rana, who owned the building outside Dhaka, and 33 other people pleaded not guilty when the charges against them were read out in court.
Another seven who absconded will be tried in absentia, prosecutor Mizanur Rahman said.
District Judge SM Kuddus Zaman said the trial will begin on September 18 over what has become known as the country’s worst industrial disaster.
Those found guilty of murder could face the death penalty as a police report submitted to the court called the deaths a “mass killing”.
About 2,500 people were injured in the April 2013 disaster.
The defendants include Rana’s parents and the owners of the five factories housed in the building.
Investigators from Bangladesh’s Criminal Investigation Department said the change from culpable homicide charges came after the investigation found Rana, his staff and the management of the five factories had allegedly forced the workers to enter the building the day of the collapse despite their unwillingness to work after the complex developed major cracks a day before.
The collapse highlighted the grim conditions in Bangladesh’s garment industry.
Low wages in the south Asian country have led global brands and retailers to prefer Bangladesh over China, the leading apparel exporter in the world.
Bangladesh has the world’s second-largest garment industry with nearly 4,000 factories employing about four million workers, mostly women.
The building collapse triggered uproar at home and abroad, with a call for reforms in a sector that helps Bangladesh earn more than £15 billion a year from exports, mainly to the United States and Europe.