Aim Herbal mouthwashes, such as persica (Salvadora persica, mint and yarrow extracts) and miswak extract have been shown to decrease gingival inflammation and plaque accumulation. The aim of this study was to compare the antimicrobial activities of persica and miswak extract with the conventional mouthwash chlorhexidine against Streptococcus salivarius, Streptococcus sanguis, Lactobacillus vulgaris and Candida albicans. Materials and methods In this in vitro study, blood-agar culture (Merk, Germany) was used to grow the streptococcus strains, saburd-dextrose culture (Merk, Germany) was used to grow C. albicans and MRS-agar was used to grow L. vulgaris. Various concentrations of these substances (0.1, 0.05 and 0.025% of miswak extract, 0.1, 0.05, 0.025 and 0.0125% of persica, 0.2, 0.1, 0.05 and 0.025% of chlorhexidine) were added to paper disks, separately, inserted into culture plates and transferred into the incubator. The inhibition zone around each disk was measured after 24 hours and the data was analyzed by the Kruskal-Wallis test. Results Chlorhexidine possessed antibacterial activity at all concentrations tested. It was more effective than persica and miswak at all concentrations on S. salivarius (p = 0.022 for 0.1%, 0.009 for 0.05 and 0.025%). It had greater effect than the other two tested material on S.sanguis only at concentration 0.01%. Chlorhexidine was the most effective against S.salivarius; persica was the most effective against Lactobacillus (p = 0.005) and the least effective against S. salivarius; and miswak extract was the most effective against S. salivarius and S. sanguis at concentrations 0.1 and 0.05% (p = 0.005) and ineffective against L. vulgaris. None of these mouthwashes were effective against C. albicans. Conclusion This study revealed that chlorhexidine remains the gold standard as an antimicrobial agent, although herbalbased mouthwashes do have marginal antimicrobial activities. It is necessary to conduct more clinical and microbiological studies focusing on periodontal pathogens and anaerobic microorganisms. Clinical significance Mechanical plaque control is the main way for periodontal disease prevention and mouthrinses are used to improve its efficacy. Based on the results of this study, chlorhexidine has the most antibacterial effect and although persica mouthwash and miswak are routinely used in some Asian countries their antibacterial efficacies are suspected. How to cite this article Moeintaghavi A, Arab H, Khajekaramodini M, Hosseini R, Danesteh H, Niknami H. In vitro Antimicrobial Comparison of Chlorhexidine, Persica Mouthwash and Miswak Extract. J Contemp Dent Pract 2012;13(2):147-152.