The bus was taking passengers from Chiang Mai to Mae Sariang district in Mae Hong Son. It left the road between kilometre markers 24 and 25 between Hot and Mae Sariang.
The bus hit a motorcycle before slamming into a tree, Hot police said.
Three people, all of them females, were found dead on the road near the motorcycle, and another male died in Hot hospital, they said.
About 50 passengers were sent to the same hospital.
An initial investigation by the district police said the bus driver lost control of the vehicle and crashed into the motorcycle coming from the opposite direction before hitting the tree.
The road in the area has several curves as it passes through mountains.