This study explores the potential of Thymus ciliatus as a natural source of bioactive compounds by investigating its chemical composition and in vitro biological activities, including antioxidant, antimicrobial, antidiabetic, and anti-Alzheimer properties. The analysis of the essential oil was performed using gas chromatography-mass spectrometry revealing α-terpinenyl acetate (18.74%) and camphor (10.62%) as the major components. Antioxidant activity was evaluated using six methods. Antimicrobial activity was assessed using disc diffusion and well diffusion techniques. Antidiabetic activity was measured through a colorimetric assay, while anti-Alzheimer activity was evaluated against butyrylcholinesterase. The results demonstrated that extracts from polar and medium-polar solvents exhibited the highest antioxidant activity, followed by low-polar solvent extracts. The essential oil of T. ciliatus displayed significant antimicrobial activity, particularly against Staphylococcus aureus, Bacillus cereus, and Candida albicans. Crude extracts also showed antimicrobial activity across all tested strains. The aqueous extract exhibited the strongest antidiabetic activity against α-glucosidase (half-maximal inhibitory concentration [IC] = 2.56 ± 0.06 µg/mL), followed by the essential oil (IC = 57.11 ± 4.39 µg/mL). Furthermore, the dichloromethane extract demonstrated the highest anti-Alzheimer activity with an IC of 0.26 ± 0.20 µg/mL. Based on these results, T. ciliatus represents a promising source of bioactive substances with potential therapeutic applications.