© 2022 Har Krishan Bhalla & Sons.This study aimed to determine the volatile profile by GC-MS and antimicrobial activity of the essential oil isolated from Thymus nummularius using the microwave-assisted hydrodistillation method and morphologically characterize the glandular trichomes in leaf samples. Obtained essential oil, as well as wastewater obtained during essential oil isolation, were chemically (polyphenolic compounds, organic acids, total phenolic content, total flavonoid content and antioxidant activity) characterized. As a result of GC-MS analysis of the essential oil, different ratios of essential oil components were identified, predominantly thymol (38.91%), linalool (13.12%) and geraniol (6.51%). The antimicrobial activity results showed that T. nummularius essential oil has a broad-spectrum inhibitory effect against all target microorganisms (bacteria and yeast-like fungi). Screening of 32 polyphenolic compounds and 52 organic acids in the wastewater was performed by LC-MS/MS and the major polyphenolic compound and organic acid were determined as caffeic acid (97316.85 µg/kg) and malic acid (6898.88 mg/kg), respectively. While the total phenolic and flavonoid content of the wastewater were 35.91 mg GA/g and 34.91 mg QE/g, respectively, the antioxidant activity results were obtained using the CUPRAC, CERAC and ABTS tests were 86.28 mg TE/g, 72.85 mg TE/g and 47.88 mg TE/g. These results support that the T. nummularius, an important production area in Turkey, can be used in different industrial areas due to its rich bioactive content. In addition, it can be said that wastewater is also considered a valuable source of bioactive substances.