The incident occurred on the road between the towns of Pereira and Pretoria early on Monday afternoon.
Gunmen opened fire on the bus, killing driver Othniel Rincon, before forcing the passengers out and burning the vehicle.
Western Fleet, the company which runs the service, suspended travel between the two departments until the future security of passengers and employees can be guaranteed. The move will affect around 150 people per day.
In response to the attack, Risaralda Governor Carlos Botero called for a greater military presence on the road.