Purpose To assess the efficacy and safety of subconjunctival fluconazole treatment as an alternative therapy for severe fungal keratitis refractory to the conventional antifungal medical treatment. Design Prospective, interventional case series. Methods This study included 13 eyes of 13 patients (mean age 49.6 years, range 28 to 75) with severe fungal keratitis that did not respond to initial therapy with topical and intravenous fluconazole plus oral itraconazole. All patients were injected with subconjunctival fluconazole 2%, up to 1.0 mL twice daily, for at least 5 days. If necessary, therapy was continued once daily for a maximum of 14 days after 5 days of injections. Results Six eyes were successfully treated after 5 days of subconjunctival injections of fluconazole. Seven patients required repeated injections for more than 5 days, but one of them finally underwent evisceration. The final visual acuity depended on the location of the remaining scar; in four cases, corneal transplantation was necessary. Neither local nor systemic toxic side effects were observed. Conclusion Subconjunctival fluconazole could be effective for treatment of severe fungal keratitis and could be very useful to avoid surgical intervention at an acute stage of this infection. Although the efficacy of the various doses cannot be determined from this series, the dose used in this study seems to be safe and effective for fungal corneal ulcer with hypopyon.