Purpose: To report the outcomes of primary transconjunctival 23-gauge (23-G) vitrectomy in the diagnosis and treatment of presumed endogenous fungal endophthalmitis (EFE). Methods: Retrospective analysis of patients with EFE who underwent diagnostic transconjunctival 23-G vitrectomy at a tertiary referral center. Results: Nineteen eyes of 15 patients with EFE were included in the study. Four patients had bilateral and 11 patients unilateral disease. Sixteen eyes of 15 patients underwent 23-G vitrectomy to confirm the diagnosis using vitreous culture, polymerase chain reaction, and histopathologic examinations. All affected eyes were treated with intravitreal amphotericin B 5 mu g/0.1 mL. Fourteen patients received additional systemic antifungal therapy. Diagnostic 23-G vitrectomy confirmed the diagnosis of EFE in 75% of the eyes (12/16). Candida was found to be a causative agent in 62.5% and Aspergillus in 12.5% of the eyes. Retinal detachment was the most common complication (42% of eyes). Conclusions: EFE can be easily confirmed using primary 23-G vitrectomy.