Background: Berberine and Nano-curcumin are two herbal medicines with strong anti-cancer effects on tumor cells, but low toxicity on normal cells, when used alone. Breast cancer is known as the most common cancer in women and second deadly one. In this study, we evaluated the cytotoxicity effects of combination Berberine and Nano-curcumin in breast cancer cell line to see whether they have further synergism cytotoxicity on MCF-7 breast cancer cell line. Methods: The cytotoxicity effects of Berberine and Nano-curcumin alone and in combination, were evaluated in MCF-7 cell lines using MTT cytotoxicity test. Statistical analysis is done through one-way ANOVA and Tukey multiple range tests. Results: Analyzing results of this study showed that cytotoxicity of Nano-curcumin was higher than Berberine in a dose-dependent manner. The IC50 of combination Berberine and Nano-curcumin was lower and showed higher cytotoxicity in MCF-7 cells compared with the time we use each of these drugs alone. Conclusion: In this study co-treatment of Berberine and Nano-curcumin significantly inhibited the growth of MCF-7 breast cancer cell line and resulted in synergism cytotoxicity effects. These results indicated on their potency to further combination of these two drugs with other agents and common chemotherapies to improve breast cancer outcomes.