The aim of this paper was to evaluate the antidiabetic and antioxidant activities of methanolic extracts, n-hexane, dichloromethane, ethyl acetate, butanol, aqueous subextracts and essential oils of flowers, roots, leaves and aerial parts of Thymbra spicata subsp. spicata, which has been utilized in the public medicine systems of Turkey, Greece, Egypt and Rome for the treatment of asthma and bronchitis, as well as for flavour and aroma in the food industry and protection. Quantitative determination of secondary metabolites in the most effective samples of the plant was also analysed by LC–MS/MS. Moreover, the chemical composition of essential oils of different parts of the plant was analysed via GC-FID and GC/MS. The main constituents in the flower, leaf and aerial part were found to be carvacrol (75.6%), γ-terpinene (10.5%), carvacrol (73.3%), γ-terpinene (9.5%), p-cymene (8.6%), carvacrol (76.1.8%) and p-cymene (7.3%), respectively. Quinic acid, caffeic acid, vanillic acid, naringin, hesperidin and rosmarinic acid were measured in four ethyl acetate subextracts, and rosmarinic acid was found to have the highest amount in the flower subextract with a value of 48095.1083 ng/mL. The ethyl acetate extract of flowers showed the best activity with a 326 ± 12 μg/mL IC50 value, while the standard acarbose IC50 value was 4143 ± 243 μg/mL. It was determined that the leaf ethyl acetate extract, in particular, had a very high % inhibition value on ABTS·+ (99.137 ± 0.011% inhibition) and DPPH• (41.068 ± 0.031% inhibition). It is thought that the plant, which has been used as a spice in the kitchen for centuries by the public, can be safely used due to its high antidiabetic and antioxidant effects.