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Satureja mutica is a perennial semi-bushy plant cultivated for different medicinal purposes. In this study, polyploidy induce was performed by using 0.00, 0.05, 0.10, and 0.20% (w/v) colchicine and three treatment duration of 6, 12, and 24 hours on one-week-old seedlings. Treated seedlings were transplanted to the medium culture and regenerated. After three regeneration, polyploid clones were identified by flow cytometry technique and chromosome counting. 2C DNA values in tetraploid (2n = 4x = 60) and induced octaploid (2n = 8x = 120) were 1.90 ± 0.01 pg and 3.82 ± 02 pg, respectively. The highest octaploid induction efficiency was 28% obtained by colchicine concentration of 0.05% and the treatment duration of 12 h. The morphological, physiological, and phytochemical characteristics were measured in tetraploid and octaploid plants. Most of the studied traits in octaploid plants except for stomatal density increased significantly, including leaf length and width, stem diameter, stomata size, area, density, leaf guard cell size, flavonoid, phenol, soluble sugar, and oil yield. In conclusion application of polyploidy induction in S. mutica could be used to increase the quality and quantity of secondary metabolites. It also can be introducing a new genotype with higher properties and drug effectiveness.