Background: Centaurea cyanus is an endemic and well-known herbal medicine in Iran, is an annual flowering plant in the family of Asteraceae. The flowers are the part used in modern herbal medicine and are considered to have tonic, stimulant and emmenagogue properties, with action similar to that of blessed thistle. The aim this study was to investigate the phytochemical constituents of C. cyanus extract, its antioxidant, anti-tumor and anti-bacterial activities. Methods: This experimental study was conducted from June to January of 2015 in Islamic Azad University of Varamin, Iran. At first, the phytochemical components of C. cyanus extract was analyzed using gas chromatography–mass spectrometry (GC-MS) method. Subsequently, the antibacterial potential of the extract was evaluated against 4 pathogenic bacteria including Staphylococcus aureus, Streptococcus pyogenes, Psedomonas aeroginosa and Klebsiella pnemoniae via minimum inhibitory concentration (MIC) mathod. Moreover, the anti-oxidant and anti-tumor activities of extract on colon cancer cell line (HT29) were investigate using DPPH and MTT colorimetric methods, respectively. Finally, the Bax and Bcl2 apoptosis gene expression level was analyzed by quantitative Real-time PCR technique. Results: GC-MS analysis of C. cyanus extract was shown 19 major components and the most frequent component was belonged to n-Hexadecanoic acid (36.4%) and Linoleic acid (19.3%). The maximum antibacterial activity of extract was observed on S. aureus and P. aeroginosa isolates. The antioxidant activity of the extract was 0.109±0.07 mg/ml. Moreover, the MTT results show that extract had IC50= 26.04±0.45 on HT29 cell line. The Real-time PCR results showed the expression level of Bax and Bcl2 was significantly increased and decreased respectively in colon cancer cell line (2.63±0.54 (P< 0.05), 0.38±0.72 (P< 0.05)). Conclusion: The results of this study show that the extract had significant anti-bacterial and anti-cancer effects and it appear that the extract has potential uses for pharmaceutical industries.