A study of antibacterial activity of the Diplostephium tolimense Cuatrec (Asteraceae) vegetal species, which was collected in the paramo of Tolima, is presented. This species, as many others of this ecosystem, has had few chemical and pharmacological studies. The simplest fraction with the largest activity against Staphylococcus aureus (ATCC 25923), is obtained starting from a raw ethanol extract and by means of bioassay guided partition. Parallelly, phytochemical analyses to the portions of greater bioactivity, obtained during the whole process, are carried out. It is observed that antibacterial action increases as the chemical simplicity increases and that the presence of terpenes and flavonoids seems to be related with this action. This is the first report, so far well-known, of in vitro antibacterial activity for D. tolimense.