Background: Hashimoto’s thyroiditis (HT) is an autoimmune disease mediated by T cells. It is characterized by the presence of thyroid autoantibodies in the serum, such as anti-thyroid peroxidase antibodies (TPO-Ab) and anti-thyroglobulin antibodies (TG-Ab). The essential oil extracted fromNigella sativaseeds is rich in bioactive substances, such as thymoquinone and cymene. Methods: Therefore, we examined the effect of essential oil fromNigella sativa(NSEO) on T cells from HT patients, especially their proliferation capacity, ability to produce cytokines, and susceptibility to apoptosis. Results: The lowest ethanol (EtOH) dilution (1:10) of NSEO significantly inhibited the proliferation of CD4+and CD8+T cells from HT patients and healthy women by affecting the percentage of dividing cells and the number of cell divisions. In addition, 1:10 and 1:50 NSEO dilutions induced cell death. Different dilutions of NSEO also reduced the concentration of IL-17A and IL-10. In healthy women, the level of IL-4 and IL-2 significantly increased in the presence of 1:10 and 1:50 NSEO dilutions. NSEO did not influence the concentration of IL-6 and IFN-γ. Conclusions: Our study demonstrates that NSEO has a strong immunomodulatory effect on the lymphocytes of HT patients.