Some food additives commonly used by humans have been recently proved to be mutagenic. It is of significant importance to evaluate their genotoxic effects, since they are frequently consumed by humans in their daily meals. In this study, we investigated the effects of monosodium glutamate (MSG), sodium benzoate (SB) and saffron on human cell lines; lung cancer (A549), breast cancer (MCF7), colon cancer (Caco-3) and normal lung (Wi38) cell line as control. Cytotoxicity of food additives was screened on multiple cell lines and examined by NR assay (Neutral Red assay). The present study focused on the cytotoxic activity of the food additives and its possible underlying mechanisms. The results showed that food additives; MSG, SB and saffron induced profound cytotoxicity in cancer cells of human colon cancer (Caco-3) (IC50 = 33.92, 15.01, 3.98 μg/ml), human breast cancer (MCF7) (IC50 = 12.79, 0.378, 10.73 μg/ml), human lung cancer cell line (A549) (IC50 = 27.37, 0.45, 2.46 μg/ml), respectively. Moreover, food additives exhibited cytotoxic activity on normal lung cell lines (Wi38) (IC50 = 4.25, 0.733, 18.14 μg/ml). These data indicated that food additives decreased cell viability in malignant and nonmalignant cells as well as confirmed the occurrence of their cytotoxic effects.