In this present study, the aerial parts of Verbascum eskisehirensis were dried in the shade and air-flowed area. The dried samples were macerated with 70% methanol in water for 24 h by three times. The biological activities of the extract was determined by in vitro methods. The antioxidant activity was evaluated in 2,2'-diphenyl-1-picrylhydrazyl (DPPH center dot) radical scavenging assay and the trolox equivalent antioxidant capacity (TEAC) was measured the ability of the extract to scavenge the stable radical cation ABTS+ (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)). The antibacterial effects against some clinical pathogens were tested by broth microdilution assay. The in vitro antiinflammatory activity was also examined by 5-Lipoxygenase (5-LOX) enzyme inhibitory test. In the same time, the phenolic composition of the extract was determined by LC-ESI-MS/MS system. According to results, the extract was found rich-in luteolin derivatives. The major compound was identified as luteolin-pentosyl-hexoside. The antioxidant activities of the extract were moderate. The extract showed weak antibacterial activity against tested bacteria strains. Salmonella typhimurium ATCC 13311 was more sensitive to the extract than the others. The extract showed weak antiinflammatory activity.