Garlic is one of the most economically important vegetables and medicinal plants. Asexual propagation of garlic has led to a critical reduction in the genetic diversity. Tetraploidization impact on morphological characteristics and enhancement of secondary compounds of garlic volatile oil was investigated in the present study. Ploidy effects were investigated following the application of 0.2, 0.4, or 0.6% (w/v) colchicine for four durations (12, 24, 36, or 48 h.) and ploidy level was identified by morphological characterization and confirmed by chromosome counting. The morphology and oil profile, determined by gas chromatography, were compared between diploid and tetraploid garlic plants. Results showed that the efficiency of tetraploid induction ranged from 8 to 26% and the highest efficiency was achieved by applying 0.6% colchicine for 24h. Confirmed tetraploid plants had larger morphological characteristics, biochemical constituents and mineral content compared to their ancestral diploid plants. However, the essential oils of the tetraploid plants and the diploid plants were found to have a substantially similar composition. Tetraploidization induction significantly increased the concentration of diallyl disulfide, allyl methyl trisulfide and diallyl trisulfide. Because the tetraploid plants contain significantly higher concentrations of sulfur components, they have unique flavor characteristics that should be of commercial interest. We concluded that polyploid induction is an attractive technique that can be used to improve garlic yield and volatile oil composition.