Purpose. Taurolidine is a broad-spectrum, nonantibiotic antimicrobial agent, not previously tested against the common causes of bacterial keratitis. This study, employing an experimental rabbit model of Staphylococcus aureus keratitis, investigated the effectiveness of topical taurolidine in reducing the number of bacteria, and its effectiveness was compared with topical ciprofloxacin, ofloxacin, and 5% cefazolin. Methods. The right corneas of all rabbits were intrastromally injected with 100 colony-forming units of Staphylococcus aureus ATCC strain 25923. The animals were divided into the following seven groups: Group 1 (6 rabbits) received taurolidine, group 2 (6 rabbits) received ciprofloxacin, group 3 (6 rabbits) received ofloxacin, group 4 (6 rabbits) received cefazolin, group 5 (5 rabbits) received polyvinylpyrrolidone ( vehicle), group 6 (4 rabbits) received sterile water, and group 7 (4 rabbits) was left untreated ( control group). The eyes were topically treated every 30 min with the above-mentioned substances from 4 to 9 h postinjection. One hour after the last drop administration (at 10 h postinjection), signs of inflammation were scored in a masked fashion by slit-lamp examination. Then, their corneas were processed. The number of colony-forming units (cfu) per cornea in all eyes was also determined. Results. All antimicrobial (taurolidine, ciprofloxacin, ofloxacin, and cefazolin) treatments significantly reduced cfu numbers and slit-lamp examination scores compared with untreated eyes, eyes that received the vehicle, or eyes with sterile water (all p values < 0.05). Regarding cfu numbers, although taurolidine therapy was significantly less effective than ciprofloxacin or ofloxacin, there was no significant difference between taurolidine and cefazolin groups. However, taurolidine had similar clinical examination scores with the other antimicrobials, while it had lower scores than the vehicle, sterile water, or untreated eyes. Conclusions. The results obtained in this study suggest that topical taurolidine is an effective, novel ocular chemotherapeutic agent for the therapy of rabbit experimental Staphylococcus aureus keratitis. This drug may be a useful and promising ocular antimicrobial.