Aims and background: The aim of the current study is to assess the antimicrobial activity of the grape seed oil (GSO) gel infused with silver nanoparticle formulated under anaerobic condition against standard chlorhexidine gel. Materials and methods: Subgingival plaque samples were obtained from individuals diagnosed with periodontitis. The plaque sample were transferred to the lab in an Eppendorf tube-containing thioglycollate broth. The samples were transferred to anaerobic chamber for incubation at room temperature for 24 hours. 20 µL of the cultured broth solution were further subcultured into the test group gel plate (GSO) and control group gel plate (chlorhexidine), the subcultured gels were of five different concentrations, namely 50, 100, 150, 200, and 250 µL. The subcultured plaque samples were transferred onto a Petri dish-containing brain heart transfusion agar. The Petri dishes were kept at 37° for 24 hours under anaerobic condition. Gas pack was placed in an anaerobic jar and evaluated after 24 hours for the number of colonies formed. Results: From the obtained results, it was observed that the number of colonies formed at 50 µL concentration in the GSO group was 275 followed by 53 colonies at 100 µL concentration. The colony count reduced to 6, 4, and 7 at 150, 200, and 250 µL concentration, respectively. The colonies formed in the control group were 4, 2, 3, 16, and 2 at 50, 100, 150, 200, 250 µL, respectively. The control group with chlorhexidine showed less number of colonies at all five concentrations. Conclusion: In conclusion, the findings highlight the noteworthy antimicrobial efficacy of GSO, particularly at higher concentrations, making it a promising alternative for managing periodontal disease. While chlorhexidine exhibited substantial microbial reduction even at lower concentrations, the natural extract of GSO offers the advantage of fewer side effects. This suggests a potential shift toward embracing GSO as a viable substitute for chlorhexidine gel in the pursuit of effective and minimally invasive periodontal care. Further research and clinical trials are warranted to solidify its role in enhancing dental health practices. Clinical significance: Grape seed oil (GSO) is a natural polyphenolic compound and has less side effects and it could be a potential agent for plaque control and management of periodontal disease. Conventional antimicrobial agents like chlorhexidine have limitations like staining of teeth and taste alterations which leads to exploration of alternative biologics.