OBJECTIVES: World Health Organization (WHO) has reported 249 million cases infected by malaria worldwide and 608 thousands deaths in 2022. Investigations for new antimalarial drugs from traditional medicine have proven to be more effective and less expensive. The medicinal plants that have been used for treatment of malaria, generally known as types of fevers in traditional medicine, can be suitable candidates for evaluating antimalarial effects. The aim of the present study was to evaluate the in vitro mechanism of antimalarial action of selected medicinal plants by inhibition test of heme detoxification (ITHD). MATERIAL AND METHODS: The methanol extract and fractions were prepared through maceration of the dry powdered plants. The ITHD method was carried in 96-wells plate and the percentage of heme polymerization inhibition was determined. The cytotoxicity of the effective plants was examined on MDBK cell line by MTT assay. Bioassay guided fractionation was performed exposing to size exclusion column chromatography and liquid-liquid fractionation and was assayed by the ITHD method for the sample with the least IC RESULTS: The methanol fraction of Viola odorata whole plant showed the most considerable results among the tested plants with IC CONCLUSION: Regarding the results of the present study and the traditional use of V. odorata for overcoming fever in Iranian traditional medicine, the final fraction of the plant could be proper candidate for further phytochemical and antimalarial studies.