Ribwort plantain (Plantago lanceolata L.), which belongs to the Plantaginaceae family, has been widely used as a herbal plant in traditional medicine across the globe. The present study aimed to investigate the biologically active substances of P. lanceolata root fractions, as well as the cytotoxic and antibacterial activities of extracts. The cytotoxic activity of ethyl acetate, dichloromethane, and n-butanol extracts of P. lanceolata root was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The P.lanceolata root extracts were also evaluated on gram-positive and negative bacteria by disc diffusion and microtiter broth dilution methods. The phytochemical content was also examined by gas chromatography-mass spectrometry. The P.lanceolata root extracts were cytotoxic; IC50 values against HCT-116 at 72 h were 168.553 μg/mL, 167.458 μg/mL, and 205.004 μg/mL for ethyl acetate, dichloromethane, and n-butanol root extracts, respectively. The dichloromethane extract of P. lanceolata root had the highest inhibitory effect against S. paratyphi (14.00±1.0 mm) at the concentration of 100 mg/mL. The minimum MIC and MBC (5 and 15 mg/mL) were observed for dichloromethane extract of P. lanceolata root against S. paratyphi. The main composition of ethyl acetate extract was 1,2-Benzenedicarboxylic acid and mono(2-ethylhexyl) ester (60.93%). The major compositions in dichloromethane and n-butanol extracts were 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (60.64%) and 2-Methyl-1-butanol (.+/-.)- (17.85%). As evidenced by the results of the present research, P. lanceolata extracts are a significant source of bioactive metabolites. Therefore, they can play a prominent role in the production of pharmaceutical materials.