Background: Persian medicine is one of the oldest and richest complementary and alternative options in the field of medicine and has a comprehensive medical system. Henna oil is recommended in Persian medicine for the treatment of numerous women’s diseases such as cervicitis. To date, henna has been used for many medical purposes, including Astringent, Bleeding, Cardioinhibitory, Hypotension, and Relaxation. Accordingly, the present study aimed to provide the formulation of a henna-oil-based vaginal suppository and examine its physicochemical and antimicrobial properties. Methods: The present study was approved and performed in accordance with the regulations of Research Council, Kerman University of Medical Sciences, Kerman, Iran, in July 2016. Different percentages of henna oil, glycerin, and gelatin, as well as henna oil and polyethylene glycol 400 and 4000, were mixed to achieve a formulation with proper appearance features and, particularly, without any oil leakage from the suppository surface. Uniformity of weight, uniformity of content, disintegration time, and dissolution test of the suppositories were evaluated. The growth-inhibiting activity of the suppositories and aqueous extract of henna was evaluated against bacteria, including the Gram-positive bacterium Gardnerella vaginalis, Neisseria gonorrhoeae, and group B streptococcus.Results: The formulations had a smooth appearance without any cracks or fractures. Disintegration times for glycero-gelatin and polyethylene glycol suppositories were 60 and 10 min, respectively. 40% of the drug was released from polyethylene glycol suppositories after 60 min, but glycero-gelatin suppositories had no releasing after 3 hours. Minimum inhibitory concentration (MIC) of suppositories and Aqueous extract were 0.4 mg/mL and 0.01 mg/ml, respectively. Conclusion: Polyethylene glycol suppositories had acceptable physicochemical properties, and the henna extract and suppositories inhibited the three studied pathogens.