Background: Porphyromonas gingivalis (P. gingivalis) is a major pathogenic bacterium that causes periodontitis, with diverse virulence factors and resistance to conventional therapies. Therefore, the search for natural antibacterial agents is important. One potential candidate is Eucheuma spinosum (E. spinosum), which contains bioactive compounds such as flavonoids, alkaloids, and terpenoids. Objectives: This study aimed to evaluate the antibacterial activity and cytotoxic effects of E. spinosum extract against P. gingivalis and BHK-21 fibroblast cells. Materials and Methods: A true experimental laboratory study was conducted using ethanol extract of E. spinosum at concentrations of 12.5%, 25%, and 50%. Antibacterial activity was assessed by disc diffusion on Mueller-Hinton agar, while cytotoxicity was evaluated by the MTT assay in BHK-21 cells. Statistical analysis was performed using Kruskal–Wallis and Mann–Whitney tests. Discussion: The results showed that E. spinosum extract exhibited significant antibacterial activity against P. gingivalis, with inhibition zones increasing in a concentration-dependent manner (10.31, 13.57, and 15.41 mm, respectively), categorized as strong inhibition. However, cytotoxicity testing revealed reduced cell viability across all concentrations (40.49%, 36.58%, and 36.53%), indicating toxic effects in accordance with ISO 10993-5. Statistical analysis confirmed significant differences between groups (p < 0.05). Conclusion: E. spinosum extract demonstrates promising antibacterial properties against P. gingivalis, but its cytotoxic effects on fibroblast cells limit its direct clinical application. Further research is required to optimize safe concentrations and improve biocompatibility for periodontal therapy.