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Peri-implant infection is a serious complication in surgical procedures involving implants. We conducted an in vitro study to determine whether the use of a fluorinated diamond-like carbon (F-DLC) coating on a titanium alloy surface can prevent peri-implant infection. After applying the F-DLC, we evaluated its antibacterial and cytotoxic properties. The coating groups, containing controlled fluorine concentrations of 5.44%, 17.43%, 24.09%, and 30%, were examined for the presence of <i>Staphylococcus aureus</i> and <i>Escherichia coli</i> according to ISO 22196 for the measurement of antibacterial activity on plastics and other nonporous surfaces. Biological toxicity was evaluated using Chinese hamster V79 cells according to ISO 10993-5 for the biological evaluation of medical devices. In the control group, populations of <i>S. aureus</i> and <i>E. coli</i> substantially increased from 2.4 × 10⁴ to (1.45 ± 1.11) × 10⁶ colony-forming units (CFUs) and from 2.54 × 10⁴ to (4.04 ± 0.44) × 10⁶ CFUs, respectively. However, no bacteria colonies were detected in any F-DLC group with a fluorine concentration of ≥ 17.43%. In the biological toxicity study, an F-DLC coating with a fluorine concentration of 30% showed a colony formation rate of 105.8 ± 24.1%, which did not differ significantly from the colony formation rate of 107.5 ± 31.1% in the nontoxic control group. An F-DLC coating on titanium alloy discs showed excellent in vitro antibacterial activity with no biological toxicity.