Purpose <p>Ocular drug delivery remains challenging due to physiological barriers that limit transcorneal permeability and cause pre-corneal drug loss, ultimately reducing the drug bioavailability in the eye. In-situ gel systems represent a promising approach for optimized therapeutic delivery and enhanced ocular bioavailability.</p> Methods <p>This study explores a stimuli-responsive in-situ gel co-loaded with Ciprofloxacin HCl (CIP) and Borneol (BOR), with BOR acting as a permeation enhancer to improve transcorneal penetration and retention of CIP in the conjunctival sac. The stimuli-responsive in situ gel was formulated and optimized using a 2³ full factorial design to systematically evaluate the effects of process variables on multiple response parameters. The formulations were extensively evaluated for clarity, pH, gelling capacity, drug release percentage, viscosity, transcorneal flux, accelerated stability, and in vivo efficacy.</p> Results <p>The in-situ gel was transparent at a pH of 6.8 and remained stable for 6 months, exhibiting acceptable physicochemical properties. The rheology of the in-situ gel under simulated tear fluid (STF) was favorable for extended ocular drug release. The optimized CIP + BOR-loaded in-situ gel demonstrated 2.5-fold higher ocular flux relative to the CIP-loaded in-situ gel, a percent drug release of 97.09 ± 1%, a gelling temperature of 34.6 ℃, and a viscosity of 240.35 mPa.s. Analytical studies differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR) confirmed that CIP and BOR were stable and safe, as indicated by the non-interacting behavior of the supporting excipients in the gel network. The hen’s egg test-chorioallantoic membrane (HET-CAM) test demonstrated that the developed in-situ gel could be safely administered in the cul-de-sac cavity, showing no irritation. The antimicrobial assay suggested that the optimal in-situ gel had superior activity compared to other groups. Histopathological evaluation of the precorneal region showed a marked decrease in corneal infiltrates and inflammation, indicating substantial recovery from the ocular infection.</p> Conclusion <p>Therefore, the stimuli-responsive in-situ gel of CIP + BOR could be a promising alternative for the treatment of bacterial conjunctivitis.</p>