Enhancing bacterial resistance to chemical antibiotics and their probabilistic side effects cause popularity of medicinal plants, so there is aninstantaneous and steady need for novel antimicrobial compounds from plants.Cucurbitamoschata(C. moschata) seeds is a native plant in Iran, which the plant has been used as an antioxidant, antiparasitic, antifungal, antiviral, and antiflammatory agent in Iran. Effect of C. moschataseeds oil on growth of the common pathogen Bacillus subtilis ATCC No. 21332 (B. subtilis) in macro-dilution Mueller-Hinton broth agar disk and agar well diffusion is described. The results of agar disk and agar well diffusion tests indicated C. moschataseeds have prevented the growth of B. subtilisand destroyed it. Also in many of samples, by increasing the concentration of C. moschata seeds, the inhibition zone increased.Partially, the widest zone of inhibition in agar diffusion (17mm) and agar well (10mm) occurred at 0.125 g/ml C. moschata seeds. Minimum inhibitory and bactericidal concentrations were 0.125 g/ml C. moschata seeds oil. There was no inhibition at 0.003, 0.007 g/ml or in dimethyl sulfoxide (DMSO). We find discrepancies in B. subtilis sensitivity to C. moschataseeds oil with greater inhibition indisk diffusion tests. Thus, the present research demonstrates the antibacterial properties of the ethnomedical plant on B. subtilis, offering to use as antibacterial supplement towards the development of new therapeutic agent.