Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (E (max)) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5-1.0, 1.0, 0.5-1.0, and 0.25 A mu g/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0-4.0, 0.5, 1.0 for A. flavus, and 0.12-0.25 A mu g/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0-16.0 A mu g/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were a parts per thousand currency sign0.03-0.5 A mu g/ml and a parts per thousand currency sign0.03 A mu g/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (E (max) = 0.63 h(-1), r (2) = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (E (max) = 0.39 h(-1), r (2) = 0.87), voriconazole (E (max) = 0.35 h(-1), r (2) = 0.098), and itraconazole (E (max) = 0.01 h(-1), r (2) = 0.98). Overall, the antifungal susceptibility of the non-fumigatus Aspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates.