Verbascum ovalifolium Donn ex Sims is a biennial plant of the Scrophulariaceae family, predominantly distributed in Bulgaria, Russia, Greece, Turkey and Romania [1, 2]. Beside the identification of 9 iridoids by TLC in this species [3], no other phytochemical and biological analyses were performed to our knowledge. The objective of this study was to investigate the phytochemical profile and biological activity of the crude methanolic extract from the aerial parts of V. ovalifolium and its hexane, ethyl acetate, butanol and aqueous fractions. The metabolite profile was determined by RP-HPLC-DAD-ESIQTOF-MS/MS. The cytotoxicity of the extracts (25-200 μg/mL) was evaluated by MTT assay towards SK-MEL-2 human melanoma cell line. Caffeoyl quinic acids, flavonoids, catalpol-type iridoids and phenylethanoid glycosides were identified in the butanol and ethyl acetate fractions. Application of high-performance counter-current chromatography (HPCCC) led to the isolation of pure luteolin-7-O-glucoside, verbascoside, saccatoside and 6-O-(3’’-O-acetyl-2’’-O-trans-p-coumaroyl)-α-Lrhamnopyranosylcatalpol (premnacorymboside A) from the ethyl acetate fraction. Premnacorymboside A was identified for the first time in Verbascum genus. The compounds were chemically characterized by 1D-NMR (1H-NMR, 13C-NMR) and 2D-NMR (COSY, HSQC, HMBC and NOESY) analyses. The viability of SK-MEL-2 melanoma cells treated with 200 μg/mL of each extract was significantly reduced between 82.52 ± 7.51% and 34.48 ± 1.15% compared to non-treated cells (p<0.001). The IC50 values for the butanol and hexane fractions were 103.77 ± 28.39 μg/mL and 160.35 ± 6.91 μg/mL, respectively. These preliminary results highlight the need for further biological investigations in order to identify the compounds responsible for the cytotoxic activity.