Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities. Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth. However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization. In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental Pseudomonas aeruginosa and Staphylococcus aureus keratitis in rabbits. The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes. Rufloxacin delivered by the polysaccharide reduced P. aeruginosa and S. aureus in the cornea at a higher rate than that obtained by rufloxacin alone. In particular, use of the polysaccharide allowed a substantial reduction of S. aureus in the cornea to be achieved even when the time interval between drug administrations was extended. These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs. The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis.