To determine the short- and long-term antibacterial properties of a novel biomedical titanium alloy to ensure excellent biocompatibility of the TiN/Ag multilayers loaded with different doses of Ag+. First, nanosized TiN/Ag multilayers were accumulated onto titanium alloy (Ti-6Al-4V) substrates via multi-arc ion plating. Then, the multilayers were implanted with different doses of silver ions (1×1017 ions/cm², 1×1018 ions/cm², 5×1016 ions/cm², and 5×1017 ions/cm²). Both short- and long-term antibacterial properties against Streptococcus mutans and Staphylococcus aureus were assessed via unique methods. Additionally, the response and behaviors of MC3T3-E1 and L929 cells on the different surfaces were evaluated by a variety of methods through comparison to a normal matched substrate (Ti-6Al-4V). In Vitro and In Vivo analyses revealed that the multilayers containing different doses of Ag ions effectively prevented bacterial adhesion and eliminated the majority of adhered bacteria in the initial period. In addition, the antibacterial activity of each TiN/Ag group improved with time, with the antibacterial rate (Ra) ultimately reaching 99% (antibacterial activity: 1 × 1018 ions/cm² > 5 × 1017 ions/cm² > 1 × 1017 ions/cm² > 5 × 1016 ions/cm²). All of the samples loaded with Ag+ exhibited good compatibility, as well as higher cell proliferation and lower apoptosis than the pure Ti-6Al-4V substrates. Considering both bacteriostasis and biocompatibility, 1 × 1017 ions/cm² and 5 × 1017 ions/cm² are the recommended doses for orthopedic and dental implants. The results indicate that all of the samples loaded with Ag+ possess excellent biocompatibility and antibacterial activity against common bacteria that cause implantation infection. The samples loaded with Ag+ can be implanted into soft and hard growing tissues to greatly improve the survival rate of orthopedic and dental implants.