The current treatment of osteosarcoma consists of chemotherapy and surgical removal, but relapse and metastasis often occur. Here tumor stem cells have an important role. They can be selectively grown from the population of tumor cells in non-adherent conditions of three-dimensional culture. Ascorbic acid is a well-known antioxidant, but under certain conditions it acts as a pro-oxidant. Research has shown that thanks to some other properties, it has a selective negative effect on the vitality of various tumor cells. The aim of this research was to test the effect of ascorbic acid treatment on the subpopulation of stem cells in spheroid culture in the presence and without the presence of FGF2 (fibroblast growth factor 2). Over 28 days, I monitored how treatment with two concentrations of ascorbic acid affects the number and size of spheroids in the presence and absence of FGF2. In addition, I tested how a 24-hour treatment with ascorbic acid affects the proportion of apoptotic cells in an adherent osteosarcoma culture. The results showed that ascorbic acid treatment has a negative effect on the number and size of spheroids and that the presence of FGF2 does not interfere with this effect. Treatment with 10 mM ascorbic acid for 24 hours significantly increased the proportion of cells that started apoptosis. The results, therefore, show that ascorbic acid has a negative effect on osteosarcoma stem cells and that the effect is independent of FGF2 presence.