The present investigation aimed to assess the antibacterial, antioxidant, anticholinesterase, and antiproliferative properties of Tribulus terrestris L. samples obtained from Iraq and Turkey. Furthermore, the total contents of flavonoids and phenols were ascertained. The above-ground parts of the plant samples were extracted with ethanol and Total antioxidant status (TAS), total oxidant status (TOS) as well as oxidative stress index (OSI) were determined. Antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Candida albicans, C. krusei and C. glabrata was assessed. Also, the antiproliferative activity was tested against the lung cancer cell line. The results showed that plant extracts were effective against microorganisms at 50-200 µg/mL concentrations. TAS values of the plant samples collected from Iraq and Türkiye were determined as 7.703±0.246 mmol/L and 6.992±0.216 mmol/L, TOS values as 15.983±0.477 μmol/L and 10.595±0.253 μmol/L, and OSI values as 0.207±0.001 and 0.152±0.005, respectively. In addition, the anti-AChE values of the samples collected from Iraq and Türkiye were 51.99±1.78 μg/mL and 74.24±2.35 μg/mL, and the anti-BChE values were 40.85±2.66 μg/mL and 64.43±2.58 μg/mL, respectively. Plant extracts showed strong effects against the A549 cancer cell line in a concentration-dependent manner. In addition, the total phenolic contents of the samples collected from Iraq and Türkiye were determined to be 90.10±3.66 mg/g and 78.24±1.51 mg/g, and the total flavonoid contents were 112.17±3.12 mg/g and 119.50±2.52 mg/g, respectively. It can be concluded that T. terrestris plants have strong biological activities that enable them to be applied as food or feed additives as well as pharmaceutical supplements.