Ebselen, an organoselenium compound, exhibits antifungal, anti-inflammatory, and antioxidant properties, making it a promising treatment for fungal keratitis. This study demonstrates that ebselen effectively inhibits Aspergillus fumigatus, reducing spore germination, mycelial growth, and biofilm formation. The antifungal effect is attributed to the disruption of the thioredoxin (Trx) system, leading to oxidative stress and cell membrane damage in fungi. Conversely, ebselen enhances the Trx system's antioxidant capacity in mammalian cells. It also suppresses inflammatory mediators (IL-1β, IL-6, TNF-α, COX-2, iNOS, and CCL2) and reduces oxidative stress markers (ROS, NO, and MDA) in fungal-stimulated macrophages. Ebselen modulates the PI3K/Akt/Nrf2 and p38 MAPK signaling pathways, improving inflammation and oxidative stress. In mouse models, ebselen reduces fungal burden, improves corneal prognosis, and decreases inflammatory mediator expression and macrophage/neutrophil infiltration. These findings suggest that ebselen is a promising treatment for fungal keratitis due to its ability to reduce fungal load and protect host tissues.Ebselen, an organoselenium compound, exhibits antifungal, anti-inflammatory, and antioxidant properties, making it a promising treatment for fungal keratitis. This study demonstrates that ebselen effectively inhibits Aspergillus fumigatus, reducing spore germination, mycelial growth, and biofilm formation. The antifungal effect is attributed to the disruption of the thioredoxin (Trx) system, leading to oxidative stress and cell membrane damage in fungi. Conversely, ebselen enhances the Trx system's antioxidant capacity in mammalian cells. It also suppresses inflammatory mediators (IL-1β, IL-6, TNF-α, COX-2, iNOS, and CCL2) and reduces oxidative stress markers (ROS, NO, and MDA) in fungal-stimulated macrophages. Ebselen modulates the PI3K/Akt/Nrf2 and p38 MAPK signaling pathways, improving inflammation and oxidative stress. In mouse models, ebselen reduces fungal burden, improves corneal prognosis, and decreases inflammatory mediator expression and macrophage/neutrophil infiltration. These findings suggest that ebselen is a promising treatment for fungal keratitis due to its ability to reduce fungal load and protect host tissues.