<p>Norfloxacin (NFX) is a broad-spectrum antibiotic with limited oral bioavailability due to its por aqueous solubility, posing challenges for effective formulation and commercialization. This study aimed to develop a novel oil-in-water (o/w) nanoemulsion system to enhance the solubility, stability, and oral absorption of NFX. The formulation utilized Tween^® 80 as the surfactant and Span™ 80 as the co-surfactant, with olive oil as the oil phase. The primary goal was to optimize the nanoemulsion for stable particle size, high zeta potential, and efficient drug entrapment. A secondary goal was to evaluate in vitro antibacterial activity againsts elected gram-positive (<i>M. luteus</i>, <i>S. aureus</i>) and gram-negative (<i>E. coli</i>, <i>P. aeruginosa</i>) bacteria at a low concentration of 25&#xa0;µg/ml. Finally, in vivo pharmacokinetics in mice assessed the oral bioavailability enhancement compared to a standard suspension. An effective NFX concentration of 5&#xa0;mg/ml was achieved in the optimized nanoemulsion. The average droplet size was 756.3 ± 5.6&#xa0;nm with good physical stability (ζ of -57 ± 35.8 mV) and uniform distribution at room temperature. The nanoemulsion exhibited strong antibacterial activity even at the lowest tested concentration. In vivo studies demonstrated significantly improved pharmacokinetics, with a 23-fold increase in AUC and prolonged half-life versus the free drug suspension. In conclusion, the optimized nanoemulsion (surfactant: co-surfactant: drug ratio of 4:1:1) markedly improved NFX solubility, dissolution, and bioavailability, likely via enhanced lymphatic uptake. These results align with predictive modeling using the desirability function in Design-Expert^® (v25.0.0).</p> Graphical Abstract <p></p>