In this study, we created ciprofloxacin.HCl (CIP)-encapsulated poly(DL-lactide-co-glycolide) (PLGA) microspheres by the solvent evaporation method. Their antibacterial activity was evaluated with pathogenic microorganisms in vitro and in vivo. Since the half-life of CIP in the blood stream is short, sustained-release properties of microspheres may provide enhanced antibacterial activity. CIP-encapsulated microspheres of PLGA were prepared by the O/O method. CIP-encapsulated PLGA microspheres showed spherical shapes under a scanning electron microscope (SEM), and their particle sizes ranged from 10 to 50 microm. In an in vitro drug release study, CIP was continuously released over 3 weeks from the microspheres, and a burst effect was observed for the first 3 days. In the in vitro antibacterial activity test, CIP-microspheres showed lower antibacterial activity compared to free CIP because of their sustained-release properties, while empty microspheres did not affect the growth of microorganisms. In the in vivo antibacterial activity test, the number of microorganisms following treatment with CIP-encapsulated microspheres was significantly lower than after treatment with free CIP at 5 days postinjection. These results suggest that encapsulated CIP keeps its antibacterial activity after microencapsulation.