Tribulus terrestris L. is an herbaceous plant belonging to the Zygophyllaceae family that is rich in diosgenin. It has anti-diabetes, anti-obesity, anti-Alzheimer, anti-inflammation, anti-leukemia, and anti-cancer activities and is a precursor of steroidal drugs. Various parts of medicinal plants have different potentials to synthesize natural compounds, so it is necessary to know which part contains higher amounts of natural compounds. Therefore, this research examined various organs of T. terrestris for their phytochemical composition and antioxidant and antibacterial properties. The highest levels of total phenolic and flavonoid content [per g of plant dry weight (DW)] were found in the leaves with 3.18 mg of gallic acid and 4.69 mg of rutin. The analysis of antioxidant activity revealed that the leaves and fruits had the highest and lowest activities (81.46 and 21.73 mu mol Fe/g DW, respectively). The high-performance liquid chromatography-photodiode array (HPLC-PDA) analysis showed vanillic acid, rutin, rosmarinic acid, and chlorogenic acid as the main phenolic acids. Based on the results, diosgenin amounts varied from 1.65 to 7.78 mg/g DW, and the highest and lowest values were observed in fruits and stems, respectively. The leaf extracts exhibited outstanding antimicrobial activity against E. coli, S. aureus, and C. albicans. Overall, the leaves outperformed other organs, considering total phenol content, total flavonoid content, antioxidant activity, and antimicrobial activity. Although the fruits had higher diosgenin content, there was no significant difference between the leaves and fruits regarding this trait. Therefore, the leaves and fruits could be used as raw materials in the pharmaceutical industry.