Objective: This study was aimed to investigate the in vitro antioxidant, antimicrobial, cytotoxicity, and enzyme inhibition activities of Tragopogon porrifolius and Polygonum cognatum which are naturally grown and consumed intensively by people in Sivas, Turkey. Methods: Plant materials were extracted with aqueous ethanol by maceration method. The components of the extracts were determined using the Gas Chromatography Mass Spectrometry. Antimicrobial, cytotoxic and enzyme inhibition activities of the extracts were investigated by micro dilution, XTT assay and 96-micro-well plate methods, respectively. The antioxidant activity evaluated using the DPPH radical scavenging, thiobarbituric acid and reducing power methods. The total phenol and total flavonoid content was also examined. Results: GC–MS analysis revealed the presence of 31 compounds inP. cognatum extract and 29 compounds in T. porrifolius extract. According to the results, T. porrifolius extract showed high level of antioxidant activity in comparison to P. Cognatum extract. T. porrifolius exhibited higher α-glucosidase inhibitory activity, and both extract showed strong α-amylase inhibition activity compared to reference drug acarbose. T. porrifolius and P. cognatum ethanolic extracts exhibited antimicrobial activity in the concentration range of 0.039–2.5 mg/ml. Both extracts also exhibited significant anticancer effect on MDA-MB-231 breast cancer cells. The IC50 values of T. porrifolius and P. cognatum extracts in MDA-MB-231 cells were determined as 0.0625 mg/mL and 0.053 mg/mL, respectively. Conclusion: Our findings demonstrated that T. porrifolius and P. cognatum ethanolic extracts have promising effect on antioxidant, antimicrobial and cytotoxic activity as well as enzyme inhibition activity, and hence further studies required to identify specific compounds responsible for these activities.