Polyelectrolyte multilayers incorporating gentamicin were fabricated using layer-by-layer deposition. The multilayers could be precisely tuned with regard to dosage, and release rate under aqueous physiological conditions could be controlled. The films were demonstrated efficacious against Staphylococcus aureus and nontoxic toward murine osteoblasts MC3T3. The films were made in a layer-by-layer process with a hereto structural architecture that represents a general strategy for incorporating charged small molecular species into polyelectrolyte multilayers without the need for any physical or chemical premodification, expanding the range of active species that can be delivered from these films while maintaining the ease of layer-by-layer fabrication process.