To assess the diagnostic utility of (1→3)-β-D-glucan (BDG) in ocular fluid of patients with fungal endophthalmitis. This prospective pilot single-center study evaluated aqueous and vitreous humor BDG levels of suspected fungal endophthalmitis, bacterial endophthalmitis, and noninfectious controls with the standard Fungitell assay and the Fungitell STAT assay. BDG levels were compared using generalized linear models followed by post-hoc pairwise comparisons. Seven fungal endophthalmitis, six bacterial endophthalmitis, and 17 non-infectious ocular samples were evaluated. Mean aqueous BDG concentrations were 204, 11.0, and 9.6 pg/ml for fungal endophthalmitis, bacterial endophthalmitis and non-infectious controls, respectively (p=0.01, fungal vs. bacterial; p=0.0005, fungal vs. non-infectious controls). Mean vitreous BDG concentrations were 165, 30.3, and 5.4 pg/ml, respectively (p=0.001 for fungal vs. bacterial; p<0.0001 for fungal vs. non-infectious controls). Mean vitreous BDG index (Fungitell STAT) values were 1.7, 0.4, and 0.3, respectively (p=0.001, fungal vs. bacterial; p=0.0004, fungal vs. non-infectious controls). The Pearson correlation between BDG levels and BDG index was high (correlation coefficient=0.99, p<0.001). Significantly elevated ocular BDG levels were found in fungal endophthalmitis compared to bacterial endophthalmitis and non-infectious controls. Our study suggests a potential utility for BDG testing in the diagnosis of fungal endophthalmitis, and a larger study is warranted.