Background: Toxoplasmosis is a serious parasitic infection caused by an obligatory intracellular parasite called Toxoplasma gondii. There is a demand for alternative anti-toxoplasmosis drugs because the available treatments for eradicating toxoplasmosis are limited. Aim of the work: This study aimed to determine the therapeutic effects of a single dose of mefloquine (MQ) on chronic toxoplasmosis, to meet the need for new anti-toxoplasmosis treatments. Materials and methods: Chronic toxoplasmosis was induced in mice under experimental conditions by the coccidian parasite T. gondii's cystogenic strain (ME49). The evaluation was based on several factors including survival rate, parasitological assessment, histopathological examination, as well as immunohistochemical evaluation of Ki-67 and NF-κB immunoreactivity. Results: MQ significantly increased the SR compared to the infected untreated group. Moreover, MQ significantly reduced the number of brain cysts (P < 0.0001) by 42.6% compared to the infected-untreated group. After treatment with MQ, fewer histopathological abnormalities were observed in the brain, liver, and spleen. Additionally, MQ significantly reduced (P < 0.05) the inflammatory score in different tissues. The administration of MQ significantly increased (P < 0.05) the Ki-67 expression in brain sections of infected mice treated with the pyrimethamine and sulfadiazine combination or MQ. Although the NF-κB expression was significantly reduced in liver sections after treatment with the pyrimethamine and sulfadiazine combination, treatment with MQ showed moderate expression in the infected-untreated tissue. Conclusion: This work demonstrated the remarkable efficacy of MQ on mice's toxoplasmosis. So, it might be used as a promising repurposed therapeutic anti-toxoplasmosis drug.