Abstract: Background and Objective: Commiphora gileadensis a medicinal plant rare species. A large amount of plant materials were needed to produce secondary metabolite under in vitro culture. Therefore, callus is used in the in vitro culture, since it can proliferate quickly and continuously provide an appropriate amount of plant which used for extracting the antimicrobial compounds from C. gileadensis. Materials and Methods: Rapid protocol for optimum callus production has been assessed to overcome limitations of the conventional propagation methods. The effect of plant growth regulator (PGR) on the regeneration of C. gileadensis was investigated for callus induction experiment using a standard MS medium with various concentrations of 6-Benzyl adenine (BA), Kinetin (Kn), 2,4-dichlorophenoxy acetic acid (2,4-D) and naphthalene acetic acid (NAA) at 0.0, 0.5, 1.0,1.5, 2.0 and 2.5 mg L–1. Results: The result showed that the maximum regeneration of callus induced the fresh and dry weight were obtained 5675±1321 and 376.7±56.9 mg, respectively on MS media containing 2 mg L–1 2,4D + 0.5 mg L–1 BA after 12 weeks. The anti-bacterial and anti-fungal activities of C. gileadensis were evaluated using the callus and ex vitro extracts, six bacterial species fungal genera were used the agar well diffusion method used of 25, 50, 75 and 100 μL methanolic or ethonlic extracts of ex vitro and callus had considerable inhibition effects on the tested bacteria and fungi. Conclusion: Callus culture technique may be an important tool to get the C. gileadensis quickly as compared to the natural growth phenomenon where it takes many years. Moreover, it’s give us an opportunity to get the active constituent without destroying the plant available in nature. The results of the present study can improve our understanding of the economic importance of C. gileadensis as activity ingredient antimicrobial agent and provided methods for its preparation.