Background & Aims: Toxoplasmosis is one of the most common parasitic infections in humans and other warm-blooded animals. One of the major problems of toxoplasmosis is its therapeutic limitation. The main treatment for this infection is the synergistic combination of pyrimethamine and sulfadiazine, though pyrimethamine is contraindicated in pregnant women owing to its teratogenicity. Considering many side effects that this parasite poses to immunocompromised individuals and pregnant women, the production of anti-TOXOPLASMA drugs with high efficacy and low side effects is therefore the main objective of TOXOPLASMA research. The goal of this study was to evaluate the cytotoxic effects of chitosan and ethanolic extract of Dracocephalum kotschyi on TOXOPLASMA GONDII tachyzoites in RH strains under in vitro condition. Materials & Methods: RH strains of TOXOPLASMA GONDII tachyzoites with the concentrations of 45, 90, and 135 mg / ml of Zarrin-Giah ethanolic extract and concentrations of 150, 300, and 600 mg / ml of chitosan, as well as components of these concentrations were evaluated. They were then compared with the positive control drug (pyrimethamine) and negative control at 24, 48, and 72 hours incubation periods, separately and also by Trypan blue staining and MTT. The lethality percentage of the extract and chitosan was assessed and compared with dead tachyzoites. All stages were evaluated by triple control. Results: Following 48-hour incubation using Trypan blue test, the concentrations of 135 mg / ml of D. kotschyi, 600 mg / ml of chitosan, and 90 extracts with 150 mg / ml of chitosan were examined. Concentrations of 500 mg / ml of control drug with lethality percentage were 50. 9, 77. 2, 88. 3, and 95. 2, and ELISA results of MTT test with ODs of 0. 098, 0. 087 0. 064, and 0. 048 showed acceptable results. After 72 hours, more than 80% of TOXOPLASMA tachyzoites were destroyed. Conclusion: Our findings revealed that chitosan extract, especially its combination with D. kotschyi extract, showed more promising results relative to the positive control drug, pyrimethamine, and the negative control. Moreover, the cytotoxic effects were maximum when these two drugs were used simultaneously.