Tour operator Intrepid Travel said 25 people -- 22 tourists and three local staff - were on board when the bus careered down an embankment outside Nairobi on Sunday night.
"Unfortunately there have been two Australian fatalities confirmed," the agency said in a statement on its Australian website.
"Other passengers have required hospital treatment for a range of injuries.
"Eighteen travellers and all staff have been transferred to a hospital in Nairobi for further assessment, with two remaining in local hospitals to be treated."
Local reports said the others on board included tourists from Britain and the United States.
Australian foreign affairs officials said consular assistance was being provided to the families of those who died and were hurt.
"It's awful beyond belief for the families of all of those involved," Australian Prime Minister Tony Abbott said.
Intrepid said its co-founder Geoff Manchester was heading to Nairobi on Monday to provide support.
