All religious insignia, including the Muslim scarf, the Christian cross and the Sikh turban, have been banned from state schools in France under a law that came into force early in September.
The two girls were expelled after school authorities failed to persuade them to abide by the law.
"In the past two months we have had several meetings with the families and with these students," Michelle Feder-Cunin, headmistress at the Jean Mace school in the city of Mulhouse, said.
The aim of the meetings had been to explain to the girls, identified only as Dounia, 12, and Khouloude, 13, the need to comply with the law.
Under the law, school authorities must first try to persuade girls to remove the headscarf and only if they persist in refusing can they be expelled.
The parents of the two students have eight days to appeal against the expulsions.
There are 72 students within the state school system in France currently breaking the law, according to education ministry figures.