The main purpose of this study is to design Cefpodoxime proxetil (CFP) loaded Eudragit RS 100 (ERS) nanoparticles to provide prolonged drug release, the enhanced oral efficacy of the drug at a lower oral dose. CFP-loaded nanoparticles are prepared by the single-emulsion solvent evaporation method with different drug:polymer ratios, F1 (CFP:ERS=1:1) and F2 (CFP:ERS=1:2). Physicochemical characterization, antibacterial activity, cytotoxicity profiles of nanoparticles are examined. F1 and F2 nanoparticles have a particle size of 236.70±3.05 and 258.87±11.32 nm, spherical shape, and a porous surface under SEM. The zeta potential values of F1 and F2 nanoparticles were 22.13±1.01 and 25.80±2.01 mV, and the encapsulation efficiencies were 34.37±3.71% and 55.78±3.52%, respectively. Since F2 nanoparticles have a smaller particle size, higher zeta potential, and encapsulation efficiency, this formulation was chosen as superior, and the subsequent experiments were continued with this formulation. The antibacterial activity of F2 nanoparticles against E.coli was found to be 2 times more effective than CFP. Cytotoxicity studies revealed that the cell viabilities in BEAS-2B and HepG2 cells were higher with the exposure to CFP nanoparticles (200 µg/ml) compared to CFP (100 µg/ml) alone. CFP nanoparticles have excellent efficacy and a cytocompatible profile in inhibiting the growth of bacteria compared to free CFP.