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This study aims to thoroughly examine the chemicals and effects of the ethanol extract from T. polium’s upper parts. We used the Soxhlet method for extraction, resulting in an extract with a significant yield of 20.6%. Qualitative analysis identified a variety of compounds, such as tannins, saponins, reducing compounds, terpenoids, quinones, and alkaloids. In quantitative analysis using the colorimetric method, we found the extract was rich in total flavonoids (20.78 mg equivalent QE/g DW extract) and total polyphenols (227.43 mg equivalent GAE/g DW extract). To assess antioxidant potential, we used the ferric reducing antioxidant power (FRAP) method, with ascorbic acid and butylated hydroxytoluene (BHT) as standards. The extract showed moderate activity in both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and FRAP methods at concentrations of 65 µg/mL and 21 mg/mL, respectively. Additionally, we tested the ethanolic extract against various bacteria using the disk diffusion technique on agar medium. The results indicated that the T. polium extract had moderate effectiveness against Gram-negative bacteria like Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 8739, as well as Gram-positive bacteria like Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633. We further investigated the composition of the ethanolic extract through LC-MS/MS analysis, establishing a detailed profile of phenolic compounds, with six flavonoids identified as the main polyphenolic constituents. This thorough evaluation provides insights into the potential therapeutic uses of T. polium.