The aim of this study was to prepare ciprofloxacin HCl (CIP)-encapsulated poly(dl-lactide-co-glycolide) (PLGA) copolymer nanoparticles and its antibacterial potential was evaluated with pathogenic bacteria, Escherichia coli (E. coli), in vitro and in vivo. CIP-encapsulated nanoparticles of PLGA were prepared by multiple emulsion solvent evaporation method. PLGA nanoparticles showed spherical shapes with particle sizes around 100-300 nm. Loading efficiency was lower than 50% (w/w) because of water-solubility properties of CIP. At drug release study, CIP showed initial burst effect for 12 h and then continuously released for 2 weeks. At in vitro antibacterial activity test, CIP-encapsulated nanoparticles showed relatively lower antibacterial activity compared to free CIP due to the sustained release characteristics of nanoparticles. However, CIP-encapsulated PLGA nanoparticles (doses: 25 mg CIP/kg of mice) effectively inhibited the growth of bacteria due to the sustained release characteristics of nanoparticles, while free CIP was less effective on the inhibition of bacterial growth. These results indicated that CIP-encapsulated PLGA nanoparticles have superior effectiveness to inhibit the growth of bacteria in vivo.