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<i>Trachyspermum ammi</i> (Apiaceae) plants have several medicinal and condimentary applications and are considered an aphrodisiac agent in Iranian Traditional Medicine. Thus, the present study aims to evaluate the effects of oil from Iranian <i>T. ammi</i> plants on the viability of spermatogonial stem cells in vitro. The essential oil of <i>T. ammi</i> fruits was extracted by hydrodistillation, and the amount of thymol was calculated by a gas-chromatography method. Spermatogonial stem cells were isolated from the testes of mice using enzyme digestion. Real-time polymerase chain reaction (RT-PCR) was applied to assess the gene expressions of promyelocytic leukemia zinc finger protein (<i>Plzf</i>), DNA-binding protein inhibitor (<i>ID-4</i>), tyrosine-protein kinase (<i>c-Kit</i>), B-cell lymphoma 2 (<i>Bcl2</i>) and Bcl2-associated X protein (<i>BAX</i>). The number and diameter of colonies were also measured in the treated cells. The amount of thymol in the oil was 130.7 &#177; 7.6 &#181;g/mL. Flow cytometry analysis showed that 92.8% of all cells expressed stimulated by retinoic acid 8 (<i>Stra8</i>), a spermatogonial stem cell marker. Expression of <i>Plzf</i> and ID-4 genes significantly increased in the treatment groups, while <i>c-Kit</i> and <i>BAX</i> decreased, and <i>Bcl2</i> increased in the presence of essential oil. The numbers and diameters of cells were also improved by the application of the plant oil. These data indicated that monoterpenes from the oil of <i>T. ammi</i> improved the quality and viability of spermatogonia cells in the cell culture.