Purpose: Compare the efficacy of treatment of pneumococcal keratitis with cholesterol, moxifloxacin, or a mixture of the two (moxifloxacin/cholesterol).Materials and Methods: New Zealand white rabbits were injected intrastromally with 106 colony-forming units (CFU) of a clinical keratitis strain of Streptococcus pneumoniae. Eyes were examined before and after treatment of topical drops every 2 hr from 25 to 47 hr post-infection (PI). Corneas were harvested to quantitate bacterial CFU, and myeloperoxidase (MPO) activity was measured at 48 hr PI. Eyes were extracted for histology. Minimal inhibitory concentrations (MICs) were determined for each compound.Results: Eyes treated with moxifloxacin/cholesterol had a significantly lower mean slit lamp examination (SLE) score than eyes treated with phosphate-buffered saline (PBS), moxifloxacin alone, or cholesterol alone (P ≤ 0.02). A significantly lower log10 CFU was recovered from corneas treated with moxifloxacin/cholesterol and moxifloxacin alone as compared to corneas of eyes treated with PBS or cholesterol alone (P < 0.01). At 48 hr PI, significantly lower MPO activity was quantitated from eyes treated with moxifloxacin/cholesterol as compared to eyes treated with cholesterol or moxifloxacin alone (P ≤ 0.046). Eyes treated with moxifloxacin/cholesterol had fewer immune cells and less corneal destruction than eyes from all other treatment groups. The MIC for moxifloxacin alone was 0.125 μg/mL, and cholesterol alone was unable to inhibit growth at any of the concentrations tested. The MIC for moxifloxacin when combined with 1% cholesterol was 0.0625 μg/mL.Conclusions: Treatment with a mixture of moxifloxacin and cholesterol significantly lowers the severity of infection caused by pneumococcal keratitis as compared to treatment with moxifloxacin alone, cholesterol alone, or PBS. This treatment mixture eradicates the bacteria in the cornea, unlike treatment with PBS or cholesterol alone. Using cholesterol with moxifloxacin as a treatment for bacterial keratitis could help lower the clinical severity of the infection.