Objective: To investigate the therapeutic role of trovafloxacin mesylate, a newer-generation fluoroquinolone with an expanded spectrum of activity, in the treatment of experimental bacterial keratitis. Methods: Susceptibility studies were performed on various strains of ocular isolates to determine the minimum inhibitory concentration (MIC) of trovafloxacin compared with ciprofloxacin and ofloxacin, using the E-test method. Pharmacokinetic studies were performed by a single topical administration of trovafloxacin to rabbit eyes with either an intact or denuded corneal epithelium. Aqueous humor, vitreous, and corneal concentrations of trovafloxacin were determined at different time points. Experimental bacterial keratitis studies were performed in rabbit eyes. Three identical studies were conducted using Staphylococcus aureus, Streptococcus pneumoniae, or Pseudomonas aeruginosa. Therapy groups included 0.5% trovafloxacin, 0.3% ciprofloxacin, 0.3% ofloxacin, and isotonic sodium chloride solution. After 12 hours of drops administration, corneas were excised, homogenized, and serially plated. The main outcome measure was quantitative bacteriologic analysis for residual colony-forming units. Results: In vitro susceptibility study findings indicated that the MIC of trovafloxacin was significantly lower than the MIC of ciprofloxacin and ofloxacin for S aureus, S pneumoniae, and Haemophilus influenzae, lower than the MIC of ciprofloxacin and ofloxacin for Staphylococcus epidermidis, and intermediate between ciprofloxacin and ofloxacin for P aeruginosa. Pharmacokinetic studies showed a significant concentration of trovafloxacin in the treated corneas, especially in eyes with a denuded epithelium. All serum samples had undetectable trovafloxacin concentrations. Experimental keratitis studies showed a statistically significant decrease of colony-forming units in trovafloxacin-treated eyes in the S aureus model and a similar decrease in the S pneumoniae and P aeruginosa models. Conclusions: Topical 0.5% trovafloxacin proved to be an effective ocular medication for the therapy of gram-positive and gram-negative keratitis. Clinical Relevance: Trovafloxacin may provide an excellent therapeutic alternative in bacterial keratitis.