Purpose: To evaluate the efficacy of topical vancomycin and povidone iodine (PI) application on methicillin-resistant Staphylococcus aureus (MRSA) keratitis model in rabbits. Methods: MRSA keratitis was induced by injecting 0.1 mL MRSA containing 1000 colony-forming units (CFU) into central cornea of right eyes of 24 New Zealand White rabbits. Animals were divided into four groups (n = 6): control (treated with balanced salt solution), 50 mg/mL topical vancomycin, 5% topical PI, and combination; examined before and after treatment, and corneal tissues were harvested for analysis at 9th hour of treatment. Results: Bacterial load was determined as: 7.63 ± 0.82 log10 CFU/g in control group, 6.95 ± 1.66 log10 CFU/g in PI group, 4.67 ± 0.77 log10 CFU/g in combination group, and 4.33 ± 0.71 log10 CFU/g in vancomycin group (p = 0.001). Median of total clinical score increased significantly from 7 [range: 5–8] to 11.5 [range: 11–15] (p = 0.001) in control group, did not change (6 [range: 5–8] to 7 [range: 5–7]; p = 0.695) in vancomycin group, increased significantly from 7 [range: 5–8] to 12.5 [range: 10–14] (p < 0.001) in PI group, increased significantly from 6.5 [range: 5–7] to 8 [range: 7–9] in combination group (p = 0.002). Post-treatment clinical scores for chemosis, conjunctival injection, iritis, hypopyon, epithelial erosion, and corneal infiltrate were significantly lower in vancomycin-treated groups compared to others (p < 0.05). In PI-treated groups, especially scores for chemosis, conjunctival injection, epithelial erosion and corneal infiltrate were significantly higher than vancomycin (p < 0.05). Conclusion: Topical vancomycin significantly inhibited bacterial growth in MRSA keratitis. However, PI was ineffective in controlling this growth; additionally, exerted toxic effect on ocular surface. When vancomycin was combined with PI, no additional increase in efficacy of treatment was detected compared to only vancomycin.