The purpose of this study was to develop and evaluate a novel dexamethasone- and tobramycin-loaded microemulsion for its potential for treating anterior segment eye infections. The microemulsion was evaluated for pH, particle size, zeta potential, light transmittance, morphology, and in vitro drug release. Sterility of the microemulsion was evaluated by direct as well as plate inoculation methods. Anti-inflammatory activity of dexamethasone, bactericidal activity of tobramycin, and cytotoxicity of the microemulsion were assessed and compared to that of the marketed eye drop suspension (Tobradex®). Histological evaluation was performed in bovine corneas to assess the safety of microemulsion in comparison to Tobradex suspension. In addition, the stability of the microemulsion was studied at 4°C, 25°C, and 40°C. The pH of the microemulsion was close to the pH of tear fluid. The microemulsion displayed an average globule size under 20 nm, with light transmittance around 95%-100%. The aseptically prepared microemulsion remained sterile for up to 14 days. The cytotoxicity of the microemulsion in bovine corneal endothelial cells was comparable to that of the Tobradex suspension. The anti-inflammatory activity of dexamethasone and the antibacterial activity of tobramycin from the microemulsion were significantly higher than those of the Tobradex suspension (P < 0.05). Histological evaluation showed an intact corneal epithelium without any signs of toxicity, and the developed microemulsion was found to be stable at 4°C and 25°C for 3 months. In conclusion, the developed microemulsion could be explored as a suitable alternative to the marketed suspension for treating anterior segment eye infections.