According to the official Bulgarian News Agency, all the victims were citizens of North Macedonia.
"According to preliminary data, a bus registered in the Republic of North Macedonia crashed on the 31st kilometre of the Struma Motorway. The accident killed 46 people," the ministry said.
It also noted that police officers, firefighters and emergency medical service personnel had arrived immediately at the scene of the accident.
Seven people, that had jumped out of the burning bus, were taken to hospital. Their condition is currently stable.
The accident reportedly occurred at 2:00 a.m. local time (00:00 GMT) near the village of Bosnek in the Pernik region in the southwest of Bulgaria.
The Bulgarian News Agency said that the bus was travelling from Turkey to North Macedonia. According to the media, there were 53 people on board.
The cause of the accident has not yet been established.
Bulgarian President Rumen Radev expressed sincere condolences to his North Macedonian counterpart, Stevo Pendarovski, and to the relatives of those injured and killed in the accident, the media reported, citing the press service of the head of state. The president also reportedly urged the responsible authorities to establish the causes of the tragedy.
European Commission President Ursula von der Leyen joined the Bulgarian leader in expressing condolences to the families of the bus-crash victims.
"My deepest condolences to the families and friends of those who perished in the tragic bus accident last night in Bulgaria. I also wish a fast recovery to those injured. In these terrible times, Europe stands in solidarity with you," she wrote on Twitter.
The Turkish Foreign Ministry also expressed condolences to the peoples of Bulgaria and North Macedonia in connection with the tragedy.
According to the media, North Macedonian Prime Minister Zoran Zaev, Foreign Minister Bujar Osmani and Health Minister Venko Filipce will travel to Bulgaria. They reportedly hope that the country's authorities will reveal the reasons for the incident and share the available information.