The genus Centaurea with an extensive background in Iranian traditional medicine represents more than 70 species in Iran that some of them are endemic to Iran. A variety of secondary metabolites has been isolated from this genus thus far. Sesquiterpene lactones and flavonoids have been reported as main compounds of C.bruguierana. Methanolic extract and different fractions of the whole fruiting samples of Centaurea bruguierana ssp.belangerana (Asteraceae) were examined for cytotoxicity against various cell lines using MTT cytotoxicity assay in order to identify active fraction(s). The chloroform and ethyl acetate fractions of plant have demonstrated significant cytotoxicity against colon adenocarcinoma and breast ductal carcinoma cell lines, which chloroform fraction, exhibited the most potent in vitro cytotoxic activity against colon adenocarcinoma cell line, and therefore, can be considered as the potential fraction through main compounds of plant against the adenocarcinoma colon cancer cell line. Moreover, only chloroform fraction was moderately active against Swiss embryo fibroblast cell lines while the other fractions were nontoxic. To summarize, the chloroform fraction of C.bruguierana demonstrated better cytotoxic activity against all of the tested cell lines compared with the other fractions; and promisingly in some cases represented moderate to very good cytotoxicity that suggests more investigations about its phytochemical properties.