NDFB militants attacked the night super bus on NH-31 at about 10 PM in Ramphalbeel area in Serfanguri police station of Kokrajhar district.
The militants stopped the bus coming from Siliguri in West Bengal on its way to Shillong in Meghalaya, sources said.
They opened fired randomly at the bus, killing five persons and critically injuring two others.
The cause of their attack on the bus is unknown, police said.
The injured have been shifted to Bongaigaon Lower Assam Hospital. The police and security forces have rushed to the spot and the area has been cordoned off, officials said.