Background: Fungal corneal ulcer is the most dangerous and challenging type of infective keratitis. Since most of the ophthalmic antifungal drops are scarce and expensive in developing countries, attempts have been made to study fungicidal property of some readily available antiseptic agents as a substitute. Povidone iodine (PI) and chlorhexidine gluconate (CHx) has been postulated to be effective against fungi. Objective: To study in vitro antifungal efficacy of PI and CHx. Methods:Fungi isolated from cases of keratomycosis were entered in a prospective study from June 2001 to March 2002. In vitro susceptibility of these fungi was tested by broth dilution method of NCCLS Standard to PI (1%, 2%, 5%, 10%) and CHx (0.04%, 0.1%, 0.2%) after 5 minutes, 1 hr, 24 hrs and 48 hrs exposure times. Results: From a total of 16 culture-proven cases of fungal keratitis, the isolated fungi were 8 Aspergillus sp, 3 Fusarium sp, 2 sterile hyphae, 1 Candida sp, 1 Drechslera sp, 1 Rhodotorula sp. PI showed 100% fungicidal effect with all tested concentrations, after 5 minutes of exposure to all fungal species.CHx. 0.1% and 0.2% after ³1 hr exposure were as effective as PI (p>0.34). The fungicidal efficacy of CHx 0.1% and 0.2% was significantly less than PI after 5 minutes (p<0.001). Conclusion:Both PI and CHx have strong in vitro fungicidal effect. The kill rate of CHx, however, is less than PI. Since in vitro efficacy of topical ophthalmic preparations is affected by multiple factors, our study provides a good idea for further in vivo investigations about this subject.