This study aimed to comprehensively assess the transcorneal permeation, antimicrobial, and antifungal activities of a nanosuspension loaded with Andrographolide, a promising herbal compound. Through a combination of in vitro and in vivo studies, the efficacy, and potential applications of the nanosuspension in ocular drug delivery were investigated. In the in vitro phase, transcorneal permeation studies were conducted using Franz diffusion cells with excised rabbit corneas. The nanosuspension demonstrated significantly enhanced permeation compared to a control formulation, indicating its ability to effectively deliver Andrographolide through the cornea and into the ocular tissues. Additionally, the nanosuspension exhibited potent antimicrobial and antifungal activities against various ocular pathogens, as determined by agar diffusion and broth microdilution assays. Building upon the promising in vitro results, in vivo studies were performed using a rabbit model. Ocular tolerability of the nanosuspension was assessed through eye observations, with no observed signs of irritation or adverse effects. Furthermore, the nanosuspension's in vivo antimicrobial and antifungal activities were evaluated by instilling the formulation into the rabbits' eyes and monitoring conjunctival congestion and inflammation. The results demonstrated a significant reduction in conjunctival congestion, highlighting the nanosuspension's potential for combating ocular infections and inflammation. The comprehensive assessment presented in this study establishes the transcorneal permeation capability of the Andrographolide-loaded nanosuspension and its remarkable antimicrobial and antifungal activities. These findings underscore the potential of the nanosuspension as an effective ocular drug delivery system for various ocular infections and inflammatory conditions. The study contributes to the development of novel therapeutic approaches in ophthalmology, aiming to improve patient outcomes and provide alternative treatment options for ocular diseases.
 Keywords: Transcorneal permeation; Nanosuspension; Andrographolide; Antimicrobial activity; Antifungal activity; Ocular drug delivery