Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol.