Leishmaniais a parasitic protozoan that tries to enter and amplify within macrophages. Macrophage cells are also immune defense cells that phagocyte many microbes like bacteria, fungi, as well as parasites likeLeishmaniaspp. However, they are unable to kill this parasite that resides in the phagosomes of contaminated macrophages and multiplies in these macrophages, leading to the destruction of contaminated macrophages and the emerging ofLeishmaniawounds. A large number of current therapies for Leishmania cure have adverse effects, or parasites have developed resistance to some of these therapies, so a better therapy for the cure of Leishmania is required. Thymoquinone is one of the Nigella Sativa ingredients with numerous biological effects, such as antioxidant as well as antimicrobial effects on a variety of microbes, namely fungi, bacteria, as well as parasites likeLeishmaniaspp. The impacts of Thymoquinone on Leishmania tropica and Leishmania infantum, as well as Leishmania-infected macrophages, were examined in this study. The impact of various Thymoquinone dosages onL. tropicaandL. infantumpromastigotes and amastigotes was examinedin vitro. Flow cytometry, as well as MTT, was also applied to examine the cytotoxic activity of Thymoquinone on promastigotes ofL. tropicaandL. infantum, as well as the incidence of apoptosis. The amastigote assay is also utilized to calculate the % of contaminated macrophages as well as the number of the present parasites in each macrophage. The percentage of macrophages contaminated with L. tropica and L. infantum amastigotes after medicating with 20 μM of Thymoquinone was 23% and 19%, respectively. Also, after medicating with 10μM of Thymoquinone, these percentages were 32% and 31%, respectively. Flow cytometry indicated that Thymoquinone caused 33.9% and 31.4% apoptosis in L. tropica and L. infantum, respectively. As determined by the promastigote assay, the inhibitory concentration (IC50) of Thymoquinone for L. tropica and L. infantum was 9.49μM and 12.66μM, respectively. The results of the promastigote and amastigote assay show that with an increase in Thymoquinone doses, its ability to kill Leishmania parasites increases, too. According to the results of the study, Thymoquinone has a potentially lethal impact onL. tropica andL. infantumpromastigotes as well as amastigotes (within leishmania contaminated macrophages).