Objective: To evaluate the anti-microsporidial effects of the active component of Nigella sativa seeds, thymoquinone, against Encephalitozoon intestinalis using an in vitro model. Methods: Anti-microsporidial effect of thymoquinone against Encephalitozoon intestinalis was evaluated by using various concentrations of thymoquinone (0, 1, 5, 10, 15, 20, 30, 35, and 40 μM) and sterile dimethyl sulfoxide. Real time PCR was used to evaluate the inhibitory effects of thymoquinone on the life cycle of Encephalitozoon intestinalis. Results: The cytotoxic effect of thymoquinone on HEK293 cell line was observed with 30, 35, and 40 μM concentrations of thymoquinone after 24, 48, and 72 hours of incubation. It was observed that 10, 15, 20, and 30 μM concentrations of thymoquinone decreased the spore density compared with the control; however, it was significant only at 30 μM. Conclusions: Thymoquinone shows potent anti-microsporidial effects against Encephalitozoon intestinalis in the in vitro model;however, the toxic concentrations of thymoquinone are also toxic to the host cells.