At least 60 others were wounded in the incident, which happened late on Monday in South Africa’s northern Limpopo province.
Police divers were searching a river that runs along the highway “for people who might have been swept away”, the regional transport department said in a statement on Tuesday.
After the crash, the tour bus rolled from a bridge on the N1 freeway into a river below, said ER24, an emergency medical care company, whose paramedics were on the scene.
“Three people were found deceased by the roadside and 16 down by the river – all were declared dead on arrival,” ER24 said in a statement.
“One patient, of the 69 passengers confirmed transported to hospital for various injuries, has since died,” it said.
It was not immediately clear how many people the tour bus was carrying.
ER24 said police were investigating the circumstances surrounding the crash.
The cause of the accident is under investigation, but heavy rains have been drenching the region in recent days, a spokesman for the regional transport department, Tidimalo Chuene, told AFP.