Poly(cyclotriphosphazene-co-4,4'-diaminodiphenyl ether) (PPO) microspheres were prepared via a precipitation polymerization method, using hexachlorocyclotriphosphazene (HCCP) and 4,4'-diaminodiphenyl ether (ODA) as monomers. Silver-loaded PPO (PPOA) microspheres were generated by the in situ loading of silver nanoparticles onto the surface by Ag+ reduction. Our results showed that PPOA microspheres were successfully prepared with a relatively uniform distribution of silver nanoparticles on microsphere surfaces. PPOA microspheres had good thermal stability and excellent antibacterial activity towards Escherichia coli and Staphylococcus aureus. Furthermore, PPOA microspheres exhibited lower cytotoxicity when compared to citrate-modified silver nanoparticles (c-Ag), and good sustained release properties. Our data indicated that polyphosphazene-based PPOA microspheres are promising antibacterial agents in the biological materials field.