"The number is now 29," said Mpumalanga province safety department spokesperson Joseph Mabuza, updating an earlier tally of 26 dead.
The bus collided with a truck near the town of Kwaggafontein, 100 kilometres east of Pretoria late on Monday night.
Three of the injured died in hospital, while eight others were still critical and another 12 slightly hurt.
The bus was travelling from Pretoria when it collided with a truck, which had swerved out of its lane, said Mabuza.
"The truck driver was trying to avoid a stationary vehicle and collided with the oncoming bus," he said. "We are not sure if the truck driver and the bus driver survived the accident."
The injured were taken to hospital in nearby KwaMhlangu. The bus company's name was not immediately available.
Mpumalanga premier's reaction
Meanwhile, Mpumalanga Premier David Mabuza expressed shock and sadness over the deaths of the passengers.
"Indeed we are shocked by such a huge number of deaths and on behalf of the provincial government I offer our deepest and most sincere condolences to the bereaved families," he said on Tuesday morning.
The premier urged motorists to be cautious and practice patience on the roads.
"Accidents are not only expensive to government but they rob families of their loved ones, breadwinners and future leaders for the country."
He also called on law enforcement agencies to help reduce road accidents so government could concentrate on service delivery.
Known as the Moloto Road after one of the towns along its way, the route is notorious for deadly collisions.
Overloaded and unroadworthy
Around 50 000 people commute to work in Pretoria daily along the narrow and potholed route, using 635 buses.
Transport vehicles using the road are often overloaded and unroadworthy and drivers are prone to speeding.
Last month, 18 people were injured in collisions on the route.
Last year, religious leaders held a prayer service for the safety of the Moloto Road. A radio station also dedicated a day to profile its dangers during which the provincial government committed to improve conditions. – Sapa