The incident happened on a motorway around 2am local time near the village of Bosnek, south-west of the capital Sofia.
Children were among the victims, ministry official Nikolai Nikolov told BTV television.
The cause of the crash and fire is being investigated. Local reports say it may have hit a curb or a guardrail.
Seven people who leapt from the burning bus were reportedly taken to hospital in Sofia and were all in a stable condition, hospital staff said.
Bulgaria's interior ministry said 45 people died, one less than the toll given earlier.
Television footage showed the bus charred and gutted by fire in the middle of the highway.
"We have an enormous tragedy here," Bulgarian interim Prime Minister Stefan Yanev told reporters.
Interior Minister Boyko Rashkov said: "The picture is terrifying, terrifying. I have never seen anything like that before."
Bulgarian investigative service chief Borislav Sarafov said four buses from a North Macedonian travel agency had entered Bulgaria late on Monday from Turkey.
"Human mistake by the driver or a technical malfunction are the two initial versions for the accident," he said.
North Macedonian Foreign Minister Bujar Osmani said the coach party was returning to the capital, Skopje, from a weekend holiday trip to the Turkish city of Istanbul, according to Reuters.
The bus reportedly had North Macedonian plates.
The area around the site of Tuesday's incident on the Struma motorway has now been sealed off.
North Macedonia's Prime Minister Zoran Zaev has already contacted his Bulgarian counterpart to discuss the tragedy.