Aim The purpose of this study was to conduct phytochemical analysis of sea buckthorn pulp oil and to evaluate the antimicrobial, anti-biofilm and antioxidant activities of its mouthwash form. Methods and Results Fatty acid composition of the sea buckthorn pulp oil was determined by GC-MS analysis, which revealed that, mono-unsaturated fatty acid, palmitoleic acid and saturated fatty acid, palmitic acid, were the major constituents. The antimicrobial and the anti-biofilm capacities of sea buckthorn pulp oil mouthwash form were evaluated against Streptococcus gordonii, Porphyromonas gingivalis, Actinomyces viscosus and Candida albicans, according to the European Norms, and the Biofilm Ring TestA (R), respectively. These activities were then compared with those of chlorhexidine and herbal mouthwashes. The sea buckthorn-based mouthwash was bactericidal against S. gordonii and P. gingivalis, bacteriostatic against A. viscosus and showed no antifungal effect. Regardless of the strains used, complete inhibition of biofilm formation was achieved. The antioxidant activity of this experimental mouthwash was also assessed by DPPH and NBT assays. Conclusion Sea buckthorn mouthwash showed anti-biofilm activities against select single and multiple oral bacterial species. Significance and Impact of the Study In this study, a mouthwash derived from sea buckthorn (Hippophae rhamnoides) pulp oil has been experimented, for the first time, in order to overcome the problem of a large number of available synthetic mouthwashes which have side effects on teeth, gums and mucous membranes. This mouthwash seemed to be a suitable alternative for a preventive agent for periodontal inflammation.