Purpose: To evaluate the safety of an ozonated solution as an antiseptic of the ocular surface prior to ophthalmic surgery. Methods: In experiment 1, a primary culture of rabbit corneal epithelium was established. Then, 0, 4 and 10 ppm ozonated solution and 1.25% povidone-iodine, respectively, were applied to confluent cells on collagen-coated filter inserts (Millicell-CM®) for 10 min followed by replacement with fresh medium. The transepithelial electrical resistance (TER), which is a good indicator of cell barrier function, was sequentially measured for 30 min. In experiment 2, adult pigmented rabbit eyes were washed with 20 ml of 4 ppm ozonated solution, 1.25% povidone-iodine solution or saline. Slitlamp examinations were performed before and after washing. Results: In experiment 1, 4 ppm ozonated solution did not change the TER as compared with the control. 10 ppm ozonated solution and 1.25% povidone-iodine similarly reduced the TER values significantly as compared with those of the control and 4 ppm ozonated solution. In experiment 2, 4 ppm ozonated solution and saline showed mild superficial punctate keratitis (SPK) in 8.3% of eyes. However, 1.25% povidone-iodine resulted in mild SPK in 17% of eyes and moderate SPK in 25% of eyes. The prevalence of SPK between two groups was significantly different (p = 0.03). Conclusion: Ozonated solution may be safe and a useful antiseptic of the ocular surface prior to ophthalmic surgery.