Background and aims: Aloysia citrodora belongs to the Verbenaceae family of plants, a well-known herbal medicine in Iran. The aim of the present study was to investigate the chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effect of A. citrodora extract against human colon cancer using real time PCR and flow cytometry methods. Materials and Methods: this experimental study was carried out in Islamic Azad University, East Tehran Branch, from March to September of 2014. At first, the A. citrodora chemical constituents were analyzed by GC/MS technique. In addition, antioxidant assay, antibacterial and anti-cancer effect was performed using DPPH, disk diffusion and MTT methods, respectively. Finally, the apoptosis gene (Bax and Bcl2) expression was performed by real time PCR and apoptotic effects was analyzed using Flow-cytometry technique. Results:  GC/MS analysis of A. citrodora extract was shown 37 major components and the most frequent component was belonged to Spathulenol (17.57%) and Caryophyllene oxide (15.15%). The antioxidant activity of the extract was IC50=0.6 ±0.03. The maximum and minimum antibacterial effects of extract were belonged to Gram negative and Gram positive bacteria, respectively. Cytotoxic results revealed that the extract have IC50= 20.1± 0.78 mg/ml against colon cell line and Real Time PCR results showed the expression level of Bax and Bcl2 was increased and decreased respectively in colon cancer cell line (3.470 ± 0.72 (P<0.05), 0.43 ± 0.35 (P< 0.05)). In addition, the flow-cytometry results indicated the 38.66 % apoptosis in colon cancer cell line. Conclusion: According to the results, it seems that A. citrodora extract has potential uses for pharmaceutical industries and it suggested that further studies were performed for A. citrodora pharmaceutical importance.