The current study aimed to analyze the chemical profile of Thymus vulgaris gathered from the Aurès zone using HPLC-DAD and GC MS methods, as well as to assess its antioxidant and antimicrobial properties. The essential oil (TVEO) and methanol extract (TVME) yielded 0.5% and 6.7%, respectively. The GC-MS analysis determined 76 volatile chemicals, with eucalyptol (11.14%), α terpinyl acetate (9.62%), (+)-2-bornanone (8.96%), 1R-α-pinene (8.74%), ledol (6.43%), and α-gurjunene (5.92%) being the main components. The HPLC-DAD profile revealed the presence of 18 compounds, with epicatechin (75.22 mg/g), caffeic acid (4.31 mg/g), luteoline-7-glucoside (2.11 mg/g), apeginin-7-glucoside (1.84 mg/g), naringenin (1.26 mg/g), and tyrosol (1.03 mg/g) being the main ingredients. TVME contained polyphenols and flavonoids (77.6 ± 0.9 μg EAG/mg and 107.36 ± 27.56 μg EQ/mg, respectively) with moderate antioxidant (IC50 = 2.94 ± 0.04 and 34.03 ± 0.13%, respectively) and antimicrobial capabilities (9.08 ± 1.66 mg). In fact, eucalyptol, (+)-2-bornanone, α-gurjunene, ledol, luteoline-7 glucoside and tyrosol were determined for the first time in T. vulgaris with high levels.