In the present work, essential oil and fatty acids and extracts obtained from aerial parts of Phlomis linearis Boiss. & Bal. were investigated for chemical composition and biological activities. The phytochemical analyses were conducted with gas chromatography-mass spectrometry/flame ionisation detector (GC-MS/FID) and liquid chromatography-mass spectromtetry (LC-MS/MS) techniques. The extracts and essential oil were studied for α -amylase and acetylcholinesterase activities with two different spectrophotometric methods. Antimicrobial activities of the extracts were investigated by microdilution. The extracts were evaluated in vitro for cytotoxic effects against cancer and normal cell lines by MTT assay. The essential oil (EO) contained α -pinene (12.5%) and β -caryophyllene (10.7%) as main compounds. Palmitic (26.5%) and nonadecanoic acids (26.6%) were determined as fatty acids. Phytochemical analysis of the extracts found phenolic acids, phlinosides, verbascoside, and flavonoids. The extracts and essential oil demonstrated poor α -amylase inhibitory activity. The best acetylcholinesterase inhibitory activity was obtained for diethly ether extract of P. linearis (67.2 ± 3.4%) at 10 mg /mL concentration. Ethyl acetate extract found to be effective against Staphlococcus aureus at a minimum inhibitory concentration (MIC) of 156.26 µg/mL. Diethyl ether extract of P. linearis was active on A549 cell lines with an IC 50 = 316 ± 4.16 µg/mL when compared with cisplatin IC 50 = 24.43 ± 0.14 µg/mL. To the best of our knowledge, the present work is the first comprehensive report on anti-acetylcholinesterase, anti-α-amylase, and antimicrobial activities, as well as cytotoxic effects of P. linearis .