There are numerous species in genus Stachys which are known for valuable and bioactive essential oils. For this study, essential oils of Stachys buttleri (SBE) and S. pinardii (SPE) were obtained by hydrodistillation method and the chemical composition of essential oils were determined by GC-MS analysis. Anticancer, antidiabetic, and antioxidant activities of essential oils were tested by MTT assay, α-glucosidase inhibitory, and ABTS radical scavenging methods, respectively. Forty-one compounds were determined in SBE (83.5%) which kolavelool (34.9%) was the main compound and diterpenoids (61.2%) were the dominant group of SBE. Thirty-three constituents were identified in SPE (90.6%) and trans-nerolidol (35.7%) and geranyl-α-terpinene (17.2%) were the most abundant compounds. SPE was characterized by its high proportion of oxygenated sesquiterpenes (39.1%), diterpenoids (21.4%), aromatics (14.8%), and sesquiterpenes (10.8%). Both essential oils showed strong anticancer activity against A549, U87MG, Ishikawa, and MCF-7 cell lines. These results indicated that SBE and SPE had potent anticancer activity. The IC₅₀ values of essential oils were between 21.33-98.3 μg/mL. Both essential oils showed moderate antioxidant and antidiabetic activities. This study is the first report on the chemical composition and biological activities of the essential oils of S. buttleri and S. pinardii.