Seven people who leapt from the burning bus were rushed to hospital in Sofia and were in stable conditions, hospital staff said. Bulgaria's interior ministry said 45 people died, one less than the toll given earlier.
The cause of the accident was unclear but the bus appeared to have hit a highway barrier either before or after it caught fire, Bulgarian officials said.
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: "People are clustered inside and are burnt to ash."
"The picture is terrifying, terrifying. I have never seen anything like that before," he told reporters at the site.
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, Reuters reported.
The accident happened on Struma highway about 45 km (28 miles) west of Sofia around 2:00 a.m. (0000 GMT).
North Macedonian Foreign Minister Bujar Osmani said the coach party was returning to Skopje from a weekend holiday trip to Istanbul.
"I am terrified. This is such a huge tragedy," North Macedonian Prime Minister Zoran Zaev told private television channel BTV.