Objectives The aim of this study was to develop and optimize levofloxacin loaded PLGA nanoparticles (LN) for pulmonary delivery employing screening and experimental design and evaluate their in-vitro and in-vivo performance. The objective was to achieve Mass Median Aerodynamic Diameter (MMAD) of LN of less than 5μm, sustain the drug release up to 120 h and a higher AUC/MIC at the site of action. Methods LN were prepared by modified emulsion solvent evaporation technique employing high speed homogenization, probe sonication and subsequent lyophilization. Key Findings The Pareto chart from Placket Burman screening design revealed that homogenization speed and amount of PLGA were found to be significant (P < 0.05). Further analysis by 3 full-factorial design revealed that F-ratio was found to be far greater than the theoretical value (P < 0.05) for each regression model. Conclusion The optimized formulation with desirability value 0.9612 showed mean particle size of 146 nm, MMAD of 4.40 μm and sustained the drug release up to 120 h in simulated lung fluid. Augmentation in Cmax (1.71-fold), AUC 0-∞ (5.46-fold), Mean Residence Time (6.64-fold) and AUC/MIC (6.21-fold) of LN through pulmonary route was found to significantly higher (P < 0.05) than levofloxacin (p. o.).