We developed a novel thermal spraying technology for a silver-containing hydroxyapatite (Ag-HA) coating with antibacterial activity to reduce the incidence of implant-associated infections. In this study, we determined the concentration of Ag ions that show antibacterial activity in fetal bovine serum (FBS) and antibacterial activity of the Ag-HA coating in FBS. The minimum inhibitory concentration (MIC) of Ag ions for several bacteria in FBS was in the range of 4.0–7.9 ppm. When 10 ppm Ag were added (as AgNO3 solution), 0.01 ppm of free Ag ions was detected. As the MIC of Ag ions approached the concentration that enabled formation of free Ag ions in FBS, the antibacterial activity of added Ag can be attributed to the free Ag ions.