Satureja mutica is a tetraploid and perennial semi-bushy plant cultivated for different medicinal purposes. To induce polyploidy, two-leafed seedlings were exposed to different concentrations (0.00, 0.05, 0.1, and 0.2 % w/v) and durations (6, 12, and 24 h) of colchicine. The seedlings were then transferred to a culture medium for recovery and propagation. After clones were prepared from each seedling, octaploid clones were identified using flow cytometry. Chromosome counting was also used to confirm flow cytometric results in tetraploid (2n = 4x = 60; 2C DNA= 1.90 ± 0.01 pg) and octaploid (2n = 8x = 120; 2C DNA= 3.82 ± 02 pg) plants. The highest polyploidy induction efficiency with 32% was related to 0.05 % colchicine and 12 h duration. The results showed that the phenotypic traits of anatomical (stomata size, leaf guard cell size), morphological (stem diameter, length, width, and leaf area), physiological (soluble sugars, phenols, and flavonoids), and phytochemical (essential oil yield, P-cymene, γ-Terpinene, α-Thujene, and α-Pinene) properties significantly increased in octaploid plants, while the density of leaf stomata decreased compared to tetraploid plants. Our results verified that octaploid induction in Satureja mutica is an effective breeding method, remarkably increasing the quantitative and qualitative characteristics, which could be used as a new genetic resource in future breeding programs.