Purpose <p>Owing to its high antibacterial efficacy and low adverse effects, local antibiotic administration has attracted attention. This in vitro study compares the antibacterial effectiveness of advanced platelet-rich fibrin plus (A-PRF+) combined with metronidazole produced under different protocols.</p> Materials and methods <p>Eight volunteers participated in this study. A 0.02% metronidazole was supplemented into A-PRF + based on four protocols concerning time (before vs. after centrifugation) and dosages (0.25 vs. 0.50 mL), yielding four groups: B25 (before centrifugation, 0.25 mL), B50 (before centrifugation, 0.50 mL), A25 (after centrifugation, 0.25 mL), and A50 (after centrifugation, 0.50 mL). Agar diffusion experiments on <i>Fusobacterium nucleatum</i> strain ATCC 25,586 were conducted to determine the bacterial efficacy of the four groups on three time points: day 2 (D2), D3 and D4. Images of the bacterial inhibition zone (BIZ) at assessment times were captured to measure BIZ dimensions using ImageJ software.</p> Results <p>No significant difference was found between the BIZs of B25 and B50; however, the BIZs of A50 were significantly higher than those of A25. BIZs of each group tend to decrease, although no significant difference was found. Two-way repeated measures ANOVA revealed a significant interaction between the timing and observation time points. Post-hoc analysis showed that at each observation time point, BIZs of A-PRF + combined with metronidazole after centrifugation were significantly higher than those before, regardless of the dosage.</p> Conclusion <p>The results of this study suggest that supplementing metronidazole into A-PRF + after centrifugation provides better antibacterial efficacy against <i>Fusobacterium nucleatum</i> than before centrifugation.</p> Clinical relevance <p>A-PRF + supplemented with metronidazole after centrifugation can exert better antibacterial efficacy against <i>Fusobacterium nucleatum</i>.</p>